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Here, we present a protocol to characterize nucleosome particles at the single-molecule level using static and time-lapse atomic force microscopy (AFM) imaging techniques. The surface functionalization method described allows for the capture of the structure and dynamics of nucleosomes in high-resolution at the nanoscale.
Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription to take place in eukaryotic cells. The dynamics of nucleosomes provides access to the DNA by replication and transcription machineries, and critically contributes to the molecular mechanisms underlying chromatin functions. Single-molecule studies such as atomic force microscopy (AFM) imaging have contributed significantly to our current understanding of the role of nucleosome structure and dynamics. The current protocol describes the steps enabling high-resolution AFM imaging techniques to study the structural and dynamic properties of nucleosomes. The protocol is illustrated by AFM data obtained for the centromere nucleosomes in which H3 histone is replaced with its counterpart centromere protein A (CENP-A). The protocol starts with the assembly of mono-nucleosomes using a continuous dilution method. The preparation of the mica substrate functionalized with aminopropyl silatrane (APS-mica) that is used for the nucleosome imaging is critical for the AFM visualization of nucleosomes described and the procedure to prepare the substrate is provided. Nucleosomes deposited on the APS-mica surface are first imaged using static AFM, which captures a snapshot of the nucleosome population. From analyses of these images, such parameters as the size of DNA wrapped around the nucleosomes can be measured and this process is also detailed. The time-lapse AFM imaging procedure in the liquid is described for the high-speed time-lapse AFM that can capture several frames of nucleosome dynamics per second. Finally, the analysis of nucleosome dynamics enabling the quantitative characterization of the dynamic processes is described and illustrated.
In eukaryotic cells, DNA is highly condensed and organized into chromosomes.1 The first level of DNA organization within a chromosome is the assembly of nucleosomes in which 147 bp of DNA is tightly wrapped around a histone octamer core.2,3 Nucleosome particles assemble on a long DNA molecule forming a chromatin array which is then organized until a highly compact chromosome unit is formed.4 The disassembly of chromatin provides the access to free DNA required by critical cellular processes such as gene transcription and genome replication, suggesting that chroma....
1. Continuous Dilution Assembly of Mono-nucleosomes
Mono-nucleosomes were first prepared for AFM imaging experiments using a continuous dilution assembly method (Figure 1). The prepared nucleosomes were then checked using discontinuous SDS-PAGE (Figure 2). A mica surface was next functionalized using APS, which captures nucleosomes at the surface while maintaining a smooth background for high-resolution imaging (Figure 3). Nucleosomes were deposited on APS-mica and were subsequently .......
The protocol described above is rather straightforward and provide highly reproducible results, although a few important issues can be emphasized. Functionalized APS-mica is a key substrate for getting reliable and reproducible results. A high stability of APS-mica is one of the important features of this substrate that allows one to prepare the imaging substrate in advance for use that can be used at least two weeks after being prepared.59,61 However, the surfac.......
Author contributions: YLL and MSD designed the project; MSD assembled nucleosomes. MSD and ZS performed AFM experiments and data analyses. All authors wrote and edited the manuscript.
....Name | Company | Catalog Number | Comments |
Plasmid pGEM3Z-601 | Addgene, Cambridge, MA | 26656 | |
PCR Primers | IDT, Coralville, IA | Custom Order | (FP) 5'- CAGTGAATTGTAATACGACTC-3' (RP) 5'-ACAGCTATGACCATGATTAC-3' |
DreamTaq polymerase | ThermoFischer Scientific, Waltham, MA | EP0701 | Catalog number for 200 units |
PCR purification kit | Qiagen, Hilden, Germany | 28104 | Catalog number for 50 units |
Tris base | Sigma-Aldrich, St. Louis, MO | 10708976001 | Catalog number for 250 g |
EDTA | ThermoFischer Scientific, Waltham, MA | 15576028 | Catalog number for 500 g |
(CENP-A/H4)2, recombinant human | EpiCypher, Durham, NC | 16-0010 | Catalog number for 50 ug |
H2A/H2B, recombinant human | EpiCypher, Durham, NC | 15-0311 | Catalog number for 50 ug |
H3 Octamer, recombinant human | EpiCypher, Durham, NC | 16-0001 | Catalog number for 50 ug |
Slide-A-Lyzer MINI Dialysis Device Kit, 10K MWCO, 0.1 mL | ThermoFischer Scientific, Waltham, MA | 69574 | Catalog number for 10 devices |
Sodium Chloride | Sigma-Aldrich, St. Louis, MO | S9888-500G | Catalog number for 500 mg |
Amicon Ultra-0.5 mL Centrifugal Filters | Millipore-sigma, Burlington, MO | UFC501008 | Catalog number for 8 devices |
HCl | Sigma-Aldrich, St. Louis, MO | 258148-25ML | Catalog number for 25 mL |
Tricine | Sigma-Aldrich, St. Louis, MO | T0377-25G | Catalog number for 25 g |
SDS | Sigma-Aldrich, St. Louis, MO | 11667289001 | Catalog number for 1 kg |
Ammonium Persulfate (AmmPS)Â | Bio-Rad, Hercules, CA | 1610700 | Catalog number for 10 g |
30% Acrylamide/Bis Solution, 37.5:1 | Bio-Rad, Hercules, CA | 1610158 | Catalog number for 500 mL |
TEMED | Bio-Rad, Hercules, CA | 1610800 | Catalog number for 5 mL |
4x Laemmli protein sample buffer for SDS-PAGE | Bio-Rad, Hercules, CA | 1610747 | Catalog number for 10 mL |
2-ME | Sigma-Aldrich, St. Louis, MO | M6250-10ML | Catalog number for 10 mL |
ageRuler Prestained Protein Ladder | ThermoFischer Scientific, Waltham, MA | 26616 | Catalog number for 500 uL |
Bio-Safeâ„¢ Coomassie Stain | Bio-Rad, Hercules, CA | 1610786 | Catalog number for 1 L |
Nonwoven cleanroom wipes: TX604 TechniCloth | TexWipe, Kernersvile, NC | TX604 | |
Muscovite Block Mica | AshevilleMica, Newport News, VA | Grade-1 | |
Aminopropyl silatrane (APS) | Synthesized as described in 22 | ||
HEPES | Sigma-Aldrich, St. Louis, MO | H4034-25G | Catalog number for 25 g |
Scotch Tape | Scotch-3M, St. Paul, MN | ||
TESPA-V2 afm probe (for static imaging) | Bruker AFM Probes, Camarillo, CA | ||
MSNL-10 afm probe (for standard time-lapse imaing) | Bruker AFM Probes, Camarillo, CA | ||
Aron Alpha Industrial Krazy Glue | Toagosei America, West Jefferson, OH | AA480 | Catalog number for 2 g tube |
MgCl2 | Sigma-Aldrich, St. Louis, MO | M8266-100G | Catalog number for 100 g |
Millex-GP Filter, 0.22 µm | Sigma-Aldrich, St. Louis, MO | SLGP05010 | Catalog number for 10 devices |
BL-AC10DS-A2 afm probe (for HS-AFM) | Olympus, Japan | ||
Compound FG-3020C-20 | FluoroTechnology Co., Ltd., Kagiya, Kasugai, Aichi, Japan | ||
Compound FS-1010S135-0.5 | FluoroTechnology Co., Ltd., Kagiya, Kasugai, Aichi, Japan | ||
MultiMode Atomic Force Microscope | Bruker-Nano/Veeco, Santa Barbara, CA | ||
High-Speed Time-Lapse Atomic Force Microsocopy | Toshio Ando, Nano-Life Science Institute, Kanazawa University, Kakuma-machi, Kanazawa, Japan |
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