Name: probing the structure and dynamics of nucleosomes using atomic force microscopy imaging
Uploaded: 2019-01-31T14:00:00
Description: Watch this Scientific Journal Video about Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging at JoVE.com

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.

1. Continuous Dilution Assembly of Mono-nucleosomes
<ol>
	<li>Generate and purify an approximately 400 bp DNA substrate that contains an off-centered Widom 601 nucleosome positioning sequence.55 
	NOTE: To limit the unwanted formation of di-nucleosomes, each &#8216;arm&#8217; flanking the positioning sequence should not exceed ~150 bp.
	<ol>
		<li>Use plasmid pGEM3Z-601 along with the designed primers and amplify the substrate DNA using PCR. For the 423 bp substrate with 122 and 154 bp arm ...

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...

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 chromatin is a highly dynamic system.5,6,7 Understanding the dynamic properties of DNA at various chromatin levels is critically important for elucidating genetic processes at the molecular level where mistakes can lead to cell death or the development of diseases such as cancer.8 A chromatin property of great importance is the dynamics of nucleosomes.9,10,11,12 The high stability of these particles has allowed for the structural characterization by crystallographic techniques.2 What these studies lack are the dynamic details of nucleosomes such as the mechanism of DNA unwrapping from the histone core; the dynamic pathway of which is required for transcription and replication processes.7,9,13,14,15,16 Furthermore, special proteins termed remodeling factors have been shown to facilitate the disassembly of nucleosomal particles17; however, the intrinsic dynamics of nucleosomes is the critical factor in this process that contributes to the entire disassembly process.14,16,18,19
Single-molecule techniques such as single-molecule fluorescence19,20,21, optical trapping (tweezers)13,18,22,23 and AFM10,14,15,16,24,25,26 have been instrumental in understanding the dynamics of nucleosomes. Among these methods, AFM benefits from several unique and attractive features. AFM allows one to visualize and characterize individual nucleosomes as well as the longer arrays27. From AFM images, important characteristics of nucleosome structure such as the length of DNA wrapped around the histone core can be measured 10,14,26,28; a parameter that is central to the characterization of nucleosome unwrapping dynamics. Past AFM studies have revealed nucleosomes to be highly dynamic systems and that DNA can spontaneously unwrap from the histone core14. The spontaneous unwrapping of DNA from nucleosomes was directly visualized by AFM operating in the time-lapse mode when the imaging is done in aqueous solutions 14,26,29.
The advent of the high-speed time-lapse AFM (HS-AFM) instrumentation made it possible to visualize the nucleosome unwrapping process at the millisecond time-scale 14,15,24. Recent HS-AFM 16,30 studies of centromere specific nucleosomes revealed several novel features of the nucleosomes compared with the canonical type. Centromere nucleosomes constitute of a centromere, a small part of the chromosome critically important for chromosome segregation31. Unlike canonical nucleosomes in bulk chromatin, the histone core of centromere nucleosomes contains CENP-A histone instead of histone H332,33. As a result of this histone substitution, DNA wrapping in centromere nucleosomes is ~120 bp instead of the ~147 bp for canonical nucleosomes; a difference that can lead to distinct morphologies of the centromere and canonical nucleosomes arrays34, suggesting that centromere chromatin undergoes higher dynamics compared with the bulk one. The novel dynamics displayed by centromere nucleosomes in HS-AFM16,30 studies exemplify the unique opportunity provided by this single-molecule technique to directly visualize the structural and dynamic properties of nucleosomes. Examples of these features will be briefly discussed and illustrated at the end of the paper. This progress was made due to the development of novel protocols for AFM imaging of nucleosomes as well as the modifications of existing methods. The goal of the protocol described here is to make these exciting advances in single-molecule AFM nucleosome studies accessible to anyone who would like to utilize these techniques in their chromatin investigations. Many of the techniques described are applicable to problems beyond the study of nucleosomes and can be used for investigations of other protein and DNA systems of interest. A few examples of such applications can be found in publications35,36,37,38,39,40,41,42,43,44,45,46,47,48,49 and prospects of AFM studies of various biomolecular systems are given in reviews29,50,51,53,54.

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		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Plasmid pGEM3Z-601</td>
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			<td height="21" style="height:21px;width:335px;">Aminopropyl silatrane (APS)</td>
			<td style="width:327px;">Synthesized as described in 22</td>
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			<td style="width:327px;">Scotch-3M, St. Paul, MN</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:335px;">TESPA-V2 afm probe (for static imaging)</td>
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			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:335px;">MSNL-10 afm probe (for standard time-lapse imaing)</td>
			<td style="width:327px;">Bruker AFM Probes, Camarillo, CA</td>
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			<td height="21" style="height:21px;width:335px;">MgCl2</td>
			<td style="width:327px;">Sigma-Aldrich, St. Louis, MO</td>
			<td style="width:119px;">M8266-100G</td>
			<td>Catalog number for 100 g</td>
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			<td height="21" style="height:21px;width:335px;">Millex-GP Filter, 0.22&#160;&#181;m</td>
			<td style="width:327px;">Sigma-Aldrich, St. Louis, MO</td>
			<td style="width:119px;">SLGP05010</td>
			<td>Catalog number for 10 devices</td>
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			<td height="21" style="height:21px;width:335px;">BL-AC10DS-A2 afm probe (for HS-AFM)</td>
			<td style="width:327px;">Olympus, Japan</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="42" style="height:42px;width:335px;">Compound FG-3020C-20&#160;</td>
			<td style="width:327px;">FluoroTechnology Co., Ltd., Kagiya, Kasugai, Aichi, Japan&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="42" style="height:42px;width:335px;">Compound FS-1010S135-0.5&#160;</td>
			<td style="width:327px;">FluoroTechnology Co., Ltd., Kagiya, Kasugai, Aichi, Japan&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:335px;">MultiMode Atomic Force Microscope</td>
			<td style="width:327px;">Bruker-Nano/Veeco, Santa Barbara, CA</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="63">
			<td height="63" style="height:63px;width:335px;">High-Speed Time-Lapse Atomic Force Microsocopy</td>
			<td style="width:327px;">Toshio Ando, Nano-Life Science Institute, Kanazawa University, Kakuma-machi, Kanazawa, Japan</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

probing the structure and dynamics of nucleosomes using atomic force microscopy imaging

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.

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 ...

Watch this Scientific Journal Video about Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging at JoVE.com

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

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 ...

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