High-Speed Time-Lapse Atomic Force Microscopy to Capture Nucleosome Dynamics

Abstract

Source: Stumme-Diers, M. P., et al., Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging. J. Vis. Exp. (2019)

In this video, we demonstrate high-speed time-lapse atomic force microscopy, AFM, imaging techniques to study the dynamics of nucleosome complexes. As the AFM tip scans across the surface of the nucleosome, it detects alterations in the nucleosome height and records the DNA unwrapping over time.

Protocol

1. Continuous Dilution Assembly of Mono-nucleosomes Generate and purify an approximately 400 bp DNA substrate that contains an off-centered Widom 601 nucleosome positioning sequence.       NOTE: To limit the unwanted formation of di-nucleosomes, each ‘arm' flanking the positioning sequence should not exceed ~150 bp. Use plasmid pGEM3Z-601 along with the designed primers and amplify the substrate DNA using PCR. For the…

Representative Results

Figure 1: Schematic of the syringe pump used for microscale nucleosome assembly. The assembly mixture is positioned to be in contact with the end of the syringe needle. As the dilution buffer is delivered by the syringe pump to the assembly mixture the concentration of NaCl is decreased, promoting nucleosome assembly. This figure is adapted from Stumme-Diers et al. <img alt="Fi…

Materials

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)	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
PageRuler Prestained Protein Ladder	ThermoFischer Scientific, Waltham, MA	26616	Catalog number for 500 uL
Bio-Safe™ 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
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
High-Speed Time-Lapse Atomic Force Microsocopy	Toshio Ando, Nano-Life Science Institute, Kanazawa University, Kakuma-machi, Kanazawa, Japan