Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

Area of Science:

• Molecular Biology
• Biophysics
• Cell Biology

Background:

• Chromatin dynamics are essential for eukaryotic DNA replication and transcription.
• Atomic force microscopy (AFM) provides critical insights into nucleosome structure and dynamics.
• Understanding nucleosome behavior is key to elucidating chromatin functions.

Purpose of the Study:

• To describe a high-resolution AFM imaging protocol for studying nucleosome structural and dynamic properties.
• To provide a detailed method for visualizing and analyzing nucleosomes, including those with CENP-A.
• To enable quantitative characterization of nucleosome dynamics.

Main Methods:

• Mono-nucleosome assembly via continuous dilution.
• Preparation of aminopropyl silatrane (APS-mica) functionalized substrate for AFM imaging.
• Static and time-lapse AFM imaging in liquid for nucleosome visualization and dynamic analysis.

Main Results:

• Demonstration of high-resolution AFM imaging for nucleosome structure.
• Measurement of DNA size wrapped around nucleosomes from static AFM images.
• Quantitative characterization of nucleosome dynamics using high-speed time-lapse AFM.

Conclusions:

• The described AFM protocol facilitates detailed structural and dynamic analysis of nucleosomes.
• This method is applicable to studying specialized nucleosomes, such as centromeric CENP-A nucleosomes.
• The protocol advances the understanding of chromatin dynamics and its role in cellular processes.