MINFLUX dissects nucleosome and compacting chromatin structures in living cells

Area of Science:

• Cell Biology
• Genetics
• Biophysics

Background:

• Chromatin structure is key for genome organization and gene regulation.
• In vitro models suggest 11-nm nucleosome polymers form 30-nm fibers, but in situ evidence is lacking.
• Existing cellular studies report disordered chains or liquid-like domains, questioning the existence of condensed fibers.

Purpose of the Study:

• To investigate the in situ existence and structure of native chromatin fibers in living cells.
• To visualize chromatin ultrastructure with high resolution and low phototoxicity.

Main Methods:

• Development of a blinking fluorescent dye for AT-rich DNA visualization.
• Application of three-dimensional (3D) MINFLUX localization microscopy for high-speed, low-phototoxicity imaging.
• Observation of chromatin fiber assembly, histone co-localization, and nucleosome folding.

Main Results:

• Visualization of native chromatin fibers (approx. 30 nm) in living cell nuclei, co-localized with histones.
• Fibers exhibit variable widths and are primarily located in the nuclear interior.
• Most observed fibers disassembled after Trichostatin A treatment, suggesting dynamic regulation.
• 3D MINFLUX revealed individual nucleosome ultrastructure, with 5-10 probes wrapping an 11-nm cylinder.

Conclusions:

• Native chromatin fibers (approx. 30 nm) do exist in living cells, particularly in AT-rich genomic regions.
• MINFLUX nanoscopy provides unprecedented insight into native chromatin ultrastructure and folding.
• This study resolves the debate on chromatin fiber existence and offers a new tool for studying genome compaction.