TY - JOUR

T1 - AFM imaging of the transcriptionally active chromatin in mammalian cells' nuclei

AU - Bairamukov, V Yu

AU - V. Filatov, M.

AU - Kovalev, R A

AU - Fedorova, N D

AU - Pantina, R A

AU - Ankudinov, A V

AU - Iashina, E G

AU - Grigoriev, S V

AU - Varfolomeeva, E Yu

N1 - Publisher Copyright: © 2022 Elsevier B.V.

PY - 2022/12

Y1 - 2022/12

N2 - BACKGROUND: 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.METHODS: Isolated nuclei were subjected to mechanical stress at 60 g and analyzed by Atomic Force Microscopy (AFM).RESULTS: 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.CONCLUSIONS: The resistance to deformation of nuclear chromatin correlates with fundamental biological processes in the cell nucleus, such as transcription, as assessed by AFM.GENERAL SIGNIFICANCE: A new outlook to studying internal nuclei structure is proposed.

AB - BACKGROUND: 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.METHODS: Isolated nuclei were subjected to mechanical stress at 60 g and analyzed by Atomic Force Microscopy (AFM).RESULTS: 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.CONCLUSIONS: The resistance to deformation of nuclear chromatin correlates with fundamental biological processes in the cell nucleus, such as transcription, as assessed by AFM.GENERAL SIGNIFICANCE: A new outlook to studying internal nuclei structure is proposed.

KW - AFM

KW - Actinomycin D

KW - Cell nucleus

KW - Chromatin

KW - Mechanical deformation

KW - Transcription

UR - http://www.scopus.com/inward/record.url?scp=85136513264&partnerID=8YFLogxK

UR - https://www.mendeley.com/catalogue/dba092da-aef9-3bc0-8842-e6adfd246d22/

U2 - 10.1016/j.bbagen.2022.130234

DO - 10.1016/j.bbagen.2022.130234

M3 - Article

C2 - 36007722

VL - 1866

JO - Biochimica et Biophysica Acta - General Subjects

JF - Biochimica et Biophysica Acta - General Subjects

SN - 0006-3002

IS - 12

M1 - 130234

ER -