Characterizing locus specific chromatin structure and dynamics with correlative conventional and super-resolution imaging in living cells

Dushyant Mehra, Santosh Adhikari, Chiranjib Banerjee, Elias M. Puchner

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

The dynamic rearrangement of chromatin is critical for gene regulation, but mapping both the spatial organization of chromatin and its dynamics remains a challenge. Many structural conformations are too small to be resolved via conventional fluorescence microscopy and the long acquisition time of super-resolution photoactivated localization microscopy (PALM) precludes the structural characterization of chromatin below the optical diffraction limit in living cells due to chromatin motion. Here we develop a correlative conventional fluorescence and PALM imaging approach to quantitatively map time-averaged chromatin structure and dynamics below the optical diffraction limit in living cells. By assigning localizations to a locus as it moves, we reliably discriminate between bound and unbound dCas9 molecules, whose mobilities overlap. Our approach accounts for changes in DNA mobility and relates local chromatin motion to larger scale domain movement. In our experimental system, we show that compacted telomeres move faster and have a higher density of bound dCas9 molecules, but the relative motion of those molecules is more restricted than in less compacted telomeres. Correlative conventional and PALM imaging therefore improves the ability to analyze the mobility and time-averaged nanoscopic structural features of locus specific chromatin with single molecule sensitivity and yields unprecedented insights across length and time scales.

Original languageEnglish (US)
Pages (from-to)E78
JournalNucleic acids research
Volume50
Issue number13
DOIs
StatePublished - Jul 22 2022

Bibliographical note

Funding Information:
This work was supported by funding from the National Institutes of Health under award number R21GM127965 and from the Mayo Graduate School of Biomedical Sciences and Mayo Foundation.

Publisher Copyright:
© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

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