Illuminating chromatin compaction in live cells and fixed tissues using SiR-DNA fluorescence lifetime

Hockings, Colin; Poudel, Chetan; Feeney, Kevin A.; Novo, Clara Lopes; Hamouda, Mehdi S.; Mela, Ioanna; Fernández‐Antorán, David; Ramirez, Pedro P. Vallejo; Rugg‐Gunn, Peter J.; Chalut, Kevin J.; Kaminski, Clemens F.; Kaminski-Schierle, Gabriele

doi:10.1101/2020.05.02.073536

Cited by 6 publications

(3 citation statements)

References 70 publications

(68 reference statements)

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“…FLIM on live interphase nuclei has shown chromatin decompaction upon chemical or radiation stimulus (Abdollahi et al 2018;Lou et al 2019;Pelicci et al 2019;Sherrard et al 2018). Live cell imaging studies have examined naive embryonic stem cells (ESCs) undergoing differentiation and showed that the presence of NANOG is essential for chromatin decompaction during pluripotency together with Rex1 downregulation (Hockings et al 2020). Furthermore, ATP depletion in ESCs leads to reduction in chromatin movement hindering transcription regulation during pluripotency (Hinde et al 2012).…”

Section: Introductionmentioning

confidence: 99%

Contribution of advanced fluorescence nano microscopy towards revealing mitotic chromosome structure

et al. 2021

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The organization of chromatin into higher-order structures and its condensation process represent one of the key challenges in structural biology. This is important for elucidating several disease states. To address this long-standing problem, development of advanced imaging methods has played an essential role in providing understanding into mitotic chromosome structure and compaction. Amongst these are two fast evolving fluorescence imaging technologies, specifically fluorescence lifetime imaging (FLIM) and super-resolution microscopy (SRM). FLIM in particular has been lacking in the application of chromosome research while SRM has been successfully applied although not widely. Both these techniques are capable of providing fluorescence imaging with nanometer information. SRM or “nanoscopy” is capable of generating images of DNA with less than 50 nm resolution while FLIM when coupled with energy transfer may provide less than 20 nm information. Here, we discuss the advantages and limitations of both methods followed by their contribution to mitotic chromosome studies. Furthermore, we highlight the future prospects of how advancements in new technologies can contribute in the field of chromosome science.

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Section: Introductionmentioning

confidence: 99%

Contribution of advanced fluorescence nano microscopy towards revealing mitotic chromosome structure

et al. 2021

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“…We therefore investigated the effect FUS condensates might have on the level of euchromatin formation, which is strongly increased by hypomethylation. We have recently shown that the dye SiR-DNA (or SiR-Hoechst) can be used to determine the level of eu/heterochromatin formation in live cells (Hockings et al, 2020; Novo et al, 2022). As SiR-DNA intercalates with DNA structures, its fluorescence lifetime is significantly affected by the level of chromatin condensation.…”

Section: Resultsmentioning

confidence: 99%

“…As SiR-DNA intercalates with DNA structures, its fluorescence lifetime is significantly affected by the level of chromatin condensation. Since euchromatin formation is associated with significant DNA decondensation, an increase in the fluorescence lifetime of SiR-DNA can be associated with an increase in euchromatin formation (Hockings et al, 2020; Novo et al, 2022). We observe that both FUS mutant cells contain more euchromatin (i.e., more decondensed DNA), with P525L-FUS cells (3.44±0.03 ns) experiencing a greater extent of DNA decondensation than R495X-FUS cells (3.43±0.04 ns) and in comparison to the GFP control (3.40±0.03 ns) and WT-FUS cells (3.41±0.03 ns) ( Figure 3C&D ).…”

Section: Resultsmentioning

confidence: 99%

Intracellular FUS protein accumulation leads to cytoskeletal, organelle and cellular homeostasis perturbations

Chung¹,

Zhou

Mela³

et al. 2022

Preprint

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The molecular mechanisms that connect the formation of aberrant cytoplasmic FUS condensates to biological malfunction are incompletely understood. Here, we develop an approach to determine the intracellular FUS viscosity in live mammalian cells and find that ALS-related mutant P525L-FUS forms the most viscous condensates and has impaired cytoskeletal mechanoproperties and increased euchromatin formation. We further show that some of the main cellular organelles, e.g., actin/tubulin, lysosomes, mitochondria, the endoplasmic reticulum, are significantly functionally/structurally impaired in the presence of FUS. These may be related to defects in the tubulin network, as the latter facilitates transport, formation, fusion and fission of organelles. We observe significant increases in lysosomal biogenesis, size and pH; moreover, intracellular FUS accumulation significantly promotes cytoplasmic-to-nuclear translocation of TFEB, i.e., the master gene for inducing autophagy. However, despite these, increased autophagy needed for protein aggregate clearance is not observed to occur. Our study reveals that the formation of highly viscous FUS condensates significantly impacts cytoskeletal/organelle function and cellular homeostasis, which are closely associated with cell ageing. This raises the intriguing question as to whether mutant FUS activates similar cell processes as those during cellular senescence.

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Satellite repeat transcripts modulate heterochromatin condensates and safeguard chromosome stability in mouse embryonic stem cells

et al. 2022

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Heterochromatin maintains genome integrity and function, and is organised into distinct nuclear domains. Some of these domains are proposed to form by phase separation through the accumulation of HP1ɑ. Mouse heterochromatin contains noncoding major satellite repeats (MSR), which are highly transcribed in mouse embryonic stem cells (ESCs). Here, we report that MSR transcripts can drive the formation of HP1ɑ droplets in vitro, and modulate heterochromatin into dynamic condensates in ESCs, contributing to the formation of large nuclear domains that are characteristic of pluripotent cells. Depleting MSR transcripts causes heterochromatin to transition into a more compact and static state. Unexpectedly, changing heterochromatin’s biophysical properties has severe consequences for ESCs, including chromosome instability and mitotic defects. These findings uncover an essential role for MSR transcripts in modulating the organisation and properties of heterochromatin to preserve genome stability. They also provide insights into the processes that could regulate phase separation and the functional consequences of disrupting the properties of heterochromatin condensates.

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Illuminating chromatin compaction in live cells and fixed tissues using SiR-DNA fluorescence lifetime

Cited by 6 publications

References 70 publications

Contribution of advanced fluorescence nano microscopy towards revealing mitotic chromosome structure

Contribution of advanced fluorescence nano microscopy towards revealing mitotic chromosome structure

Intracellular FUS protein accumulation leads to cytoskeletal, organelle and cellular homeostasis perturbations

Satellite repeat transcripts modulate heterochromatin condensates and safeguard chromosome stability in mouse embryonic stem cells

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