AFM imaging of the transcriptionally active chromatin in mammalian cells' nuclei

Nuclear rigidity is traditionally associated with lamina and densely packed heterochromatin. Actively transcribed DNA is thought to be less densely packed. Currently, approaches for direct measurements of the transcriptionally active chromatin rigidity are quite limited. Isolated nuclei were subject...

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Veröffentlicht in:Biochimica et biophysica acta. General subjects Jg. 1866; H. 12; S. 130234
Hauptverfasser: Bairamukov, V.Yu, Filatov, M.V., Kovalev, R.A., Fedorova, N.D., Pantina, R.A., Ankudinov, A.V., Iashina, E.G., Grigoriev, S.V., Varfolomeeva, E.Yu
Format: Journal Article
Sprache:Englisch
Veröffentlicht: Elsevier B.V 01.12.2022
Schlagworte:
Actinomycin D
AFM
atomic force microscopy
Cell nucleus
Chromatin
deformation
fibroblasts
heterochromatin
landscapes
mammals
Mechanical deformation
supercoiled DNA
Transcription
transcription (genetics)
Actinomycin D
AFM
Chromatin
Transcription
Cell nucleus
Mechanical deformation
ISSN:0304-4165, 1872-8006, 1872-8006
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Zusammenfassung:Nuclear rigidity is traditionally associated with lamina and densely packed heterochromatin. Actively transcribed DNA is thought to be less densely packed. Currently, approaches for direct measurements of the transcriptionally active chromatin rigidity are quite limited. Isolated nuclei were subjected to mechanical stress at 60 g and analyzed by Atomic Force Microscopy (AFM). Nuclei of the normal fibroblast cells were completely flattened under mechanical stress, whereas nuclei of the cancerous HeLa were extremely resistant. In the deformed HeLa nuclei, AFM revealed a highly-branched landscape assembled of ~400 nm closed-packed globules and their structure was changing in response to external influence. Normal and cancerous cells' isolated nuclei were strikingly different by DNA resistance to applied mechanical stress. Paradoxically, more transcriptionally active and less optically dense chromatin of the nuclei of the cancerous cells demonstrated higher physical rigidity. A high concentration of the transcription inhibitor actinomycin D led to complete flattening of HeLa nuclei, that might be related to the relaxation of supercoiled DNA tending to deformation. At a low concentration of actinomycin D, we observed the intermediary formation of stochastically distributed nanoloops and nanofilaments with different shapes but constant width ~ 180 nm. We related this phenomenon with partial DNA relaxation, while non-relaxed DNA still remained rigid. The resistance to deformation of nuclear chromatin correlates with fundamental biological processes in the cell nucleus, such as transcription, as assessed by AFM. A new outlook to studying internal nuclei structure is proposed. [Display omitted] •The method of the imaging of transcriptionally active chromatin by AFM via nuclei mechanical stress was proposed.•Nuclear DNA of normal or transcriptionally inactive cells nuclei was completely flattened under mechanical stress.•HeLa cells' nuclei were sustainable to deformation, revealing the globular higher-packed architecture of the supercoiled DNA.•Transcription inhibitor actinomycin D relaxed tensions of supercoiled DNA reducing its resistance to mechanical stress.•Our results demonstrate a relationship between nuclear mechanics and transcription.
Bibliographie:ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
ISSN:0304-4165
1872-8006
1872-8006
DOI:10.1016/j.bbagen.2022.130234