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.

The authors declare that they have no competing financial interests.

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 surface can be damaged by vapors of glue if it is used for mica mounting on the metal puck. Therefore, it is recommended to use double stick tape for the mica mounting as described in the protocol (section 2). In cases when the use of glue is necessary, for example, some users use glues for mounting of mica on metal disks for imaging in liquid, the following procedure modified from the one described in section 6.1.3 for the sample preparation for HS-AFM imaging is recommended. Mica is glued to the metal disk or other solid material and cleaved after the glue is solidified. Blow the mica with argon to remove potential vapors of the glue. Then the mica can be cleaved with a scotch tape and the APS mica working solution placed to cover the freshly cleaved mica strip. The amount of the solution depends on the mica strip size. Allow APS to react for 30 min. To minimize evaporation, run the reaction in a wet Petri dish. Use the following procedure: Soak lab wipe paper and place is at the bottom of the Petri dish. Place a 5 mm thick plastic disk on the wet wipes and put the metal disk with glued mica on it avoiding contact with water at the bottom of the Petri dish. After 30 min, remove the mica/disk assembly and thoroughly rinse the mica surface with dd water and pipette as described in section 6.1.3. The surface is ready for the sample deposition. The APS concentration needs to be adjusted in experiments with DNA, although working APS solution (1:300) described in section 2.2 should be a good starting point. In the protocol described in section 6.1.3 in which the procedure for imaging of nucleosomes with HS-AFM is described, the three-fold higher concentration of APS (1:100) was used.&#160;&#160;
The protocol described is based on mica functionalization with APS. This is the most robust, highly reproducible and simple procedure. The only disadvantage is that APS, aminopropyl silatrane is not commercially available. However, the procedure of the APS synthesis is straightforward and the protocol for its synthesis is described in detail. 59 If the synthesis procedure is problematic, commercially available reagent, aminopropyltriethoxy silane (APTES) can be used for mica functionalization (AP-mica). The same paper provides the protocol for the preparation of AP-mica using vapors of APTES. The procedure was tested and used for imaging of topologically different types of DNA 35,36,50,62,63 and various protein-DNA complexes including nucleosomes. 27,50,64 Similarly to APS-mica, AP-mica is stable over weeks and can be prepared in batches in advance; the AP-mica surface is as smooth as APS-mica and rather insensitive to the buffer composition, so the samples can be prepared in the buffer solutions with pH values up to pH10 and ionic strengths between 1 mM and 200 mM of NaCl. 59,65 The only drawback with the use of AP-mica is the possibility of aminopropyl silane to hydrolyze and assemble in aggregates on the surface during time-lapse imaging in water, although this process is rather slow with the aggregates that can be distinguished on the surface, so high-resolution images of DNA can be obtained.35 The hydrolysis of silatrane moiety is very slow, so no visible aggregates are seen during long-term imaging;26,35,56,66 , therefore, APS-mica is the preferable substrate when compared with&#160; AP-mica if imaging in water is employed. For reproducibility using APTES, the distillation of the reagent is highly recommended in the preparation of AP-mica. The protocol for the APTES distillation is described in paper 59.
AFM, as a single molecule technique, operates with nucleosome concentrations at the nanomolar range and that nucleosomes can spontaneously dissociate at such low concentrations. According to our data 25, the dissociation process occurs in dozens of minutes suggesting that the sample for AFM studies should be prepared just prior to the deposition. Some detergents such as 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) increase the nucleosome stability, so the nucleosomes increase their lifetime by several orders of magnitude 25, but the use of detergents should be done with caution. We showed in 25 that stabilization of nucleosomes is accompanied by the change of the sequence specificity, so the nucleosome even with the use of such sequence specific motif as Widom 601 template, assemble without a specificity of binding to the 601 sequence.
There are a number of important issues with the proposed method relative to other AFM sample preparation methods. First, APS-mica and AP-mica substrates are stable over weeks and can be prepared in batches in advance; the surfaces are smooth and rather insensitive to the buffer composition so the samples can be prepared in buffer solutions with pH values up to pH10 and ionic strengths between 1 mM and 200 mM of NaCl. 59,65 None of the existing methods match these properties. Second, AFM is a single molecule technique and requires a very little sample. The described protocol operates with nanoscale amounts of the preparation. Third, in time-lapse AFM studies, and the HS-AFM data acquisition, there is a significant amount of time required to generate and characterize the large data sets acquired. The methodology described here is well suited for analyzing hundreds of AFM images.
The sample preparation methodology is not limited to nucleosome type samples. Rather it is insensitive to the type of the protein-DNA complexes and has already been applied to a number proteins recognizing specific sequences on DNA, e.g. restrictions enzymes 67,68,69, single-stranded DNA binding proteins 44,45,56,70,71 along with complex protein systems involved in DNA replication 47,72 and recombination68,73. Importantly, HS-AFM has been applied to these systems to follow the dynamics of these systems.&#160; These studies make it possible to apply the developed methodology to the characterization of nucleosome arrays as proposed in and elucidate the role of such centromere specific proteins as centromere protein B (CENP-B) or C (CENP-C) in the centromere assembly and understanding their role in the development of many cancers33,34.

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.

<table border="0" cellpadding="0" cellspacing="0" style="width:1229px;" width="1229">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Plasmid pGEM3Z-601</td>
			<td style="width:327px;">Addgene, Cambridge, MA</td>
			<td style="width:119px;">26656</td>
			<td style="width:449px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">PCR Primers</td>
			<td style="width:327px;">IDT, Coralville, IA</td>
			<td style="width:119px;">Custom Order</td>
			<td>(FP) 5&#39;- CAGTGAATTGTAATACGACTC-3&#39; (RP) 5&#39;-ACAGCTATGACCATGATTAC-3&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">DreamTaq polymerase</td>
			<td style="width:327px;">ThermoFischer Scientific, Waltham, MA</td>
			<td style="width:119px;">EP0701</td>
			<td>Catalog number for 200 units</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">PCR purification kit</td>
			<td style="width:327px;">Qiagen, Hilden, Germany&#160;</td>
			<td style="width:119px;">28104</td>
			<td>Catalog number for 50 units</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Tris base</td>
			<td style="width:327px;">Sigma-Aldrich, St. Louis, MO</td>
			<td style="width:119px;">10708976001</td>
			<td>Catalog number for 250 g</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:335px;">EDTA</td>
			<td style="width:327px;">ThermoFischer Scientific, Waltham, MA</td>
			<td>15576028</td>
			<td>Catalog number for 500 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">(CENP-A/H4)2, recombinant human</td>
			<td style="width:327px;">EpiCypher, Durham, NC</td>
			<td style="width:119px;">16-0010</td>
			<td>Catalog number for 50 ug</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">H2A/H2B, recombinant human</td>
			<td style="width:327px;">EpiCypher, Durham, NC</td>
			<td style="width:119px;">15-0311</td>
			<td>Catalog number for 50 ug</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">H3 Octamer, recombinant human</td>
			<td style="width:327px;">EpiCypher, Durham, NC</td>
			<td style="width:119px;">16-0001</td>
			<td>Catalog number for 50 ug</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Slide-A-Lyzer MINI Dialysis Device Kit, 10K MWCO, 0.1 mL</td>
			<td style="width:327px;">ThermoFischer Scientific, Waltham, MA</td>
			<td style="width:119px;">69574</td>
			<td>Catalog number for 10 devices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Sodium Chloride</td>
			<td style="width:327px;">Sigma-Aldrich, St. Louis, MO</td>
			<td style="width:119px;">S9888-500G</td>
			<td>Catalog number for 500 mg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Amicon Ultra-0.5&#160;mL Centrifugal Filters&#160;</td>
			<td style="width:327px;">Millipore-sigma, Burlington, MO</td>
			<td style="width:119px;">UFC501008</td>
			<td>Catalog number for 8 devices</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">HCl</td>
			<td style="width:327px;">Sigma-Aldrich, St. Louis, MO</td>
			<td>258148-25ML</td>
			<td>Catalog number for 25 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Tricine</td>
			<td style="width:327px;">Sigma-Aldrich, St. Louis, MO</td>
			<td>T0377-25G</td>
			<td>Catalog number for 25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">SDS</td>
			<td style="width:327px;">Sigma-Aldrich, St. Louis, MO</td>
			<td style="width:119px;">11667289001</td>
			<td>Catalog number for 1 kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Ammonium Persulfate (AmmPS)&#160;</td>
			<td style="width:327px;">Bio-Rad, Hercules, CA</td>
			<td style="width:119px;">1610700</td>
			<td>Catalog number for 10 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">30% Acrylamide/Bis Solution, 37.5:1</td>
			<td style="width:327px;">Bio-Rad, Hercules, CA</td>
			<td style="width:119px;">1610158</td>
			<td>Catalog number for 500 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">TEMED</td>
			<td style="width:327px;">Bio-Rad, Hercules, CA</td>
			<td style="width:119px;">1610800</td>
			<td>Catalog number for 5 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">4x Laemmli protein sample buffer for SDS-PAGE</td>
			<td style="width:327px;">Bio-Rad, Hercules, CA</td>
			<td style="width:119px;">1610747</td>
			<td>Catalog number for 10 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">2-ME</td>
			<td style="width:327px;">Sigma-Aldrich, St. Louis, MO</td>
			<td style="width:119px;">M6250-10ML</td>
			<td>Catalog number for 10 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">ageRuler Prestained Protein Ladder&#160;</td>
			<td style="width:327px;">ThermoFischer Scientific, Waltham, MA</td>
			<td style="width:119px;">26616</td>
			<td>Catalog number for 500 uL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Bio-Safe&#8482; Coomassie Stain</td>
			<td style="width:327px;">Bio-Rad, Hercules, CA</td>
			<td style="width:119px;">1610786</td>
			<td>Catalog number for 1 L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Nonwoven cleanroom wipes: TX604 TechniCloth&#160;</td>
			<td style="width:327px;">TexWipe, Kernersvile, NC</td>
			<td style="width:119px;">TX604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Muscovite Block Mica</td>
			<td style="width:327px;">AshevilleMica, Newport News, VA</td>
			<td style="width:119px;">Grade-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Aminopropyl silatrane (APS)</td>
			<td style="width:327px;">Synthesized as described in 22</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">HEPES</td>
			<td style="width:327px;">Sigma-Aldrich, St. Louis, MO</td>
			<td style="width:119px;">H4034-25G</td>
			<td>Catalog number for 25 g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Scotch Tape</td>
			<td style="width:327px;">Scotch-3M, St. Paul, MN</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">TESPA-V2 afm probe (for static imaging)</td>
			<td style="width:327px;">Bruker AFM Probes, Camarillo, CA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<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>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Aron Alpha Industrial Krazy Glue</td>
			<td style="width:327px;">Toagosei America, West Jefferson, OH</td>
			<td style="width:119px;">AA480</td>
			<td>Catalog number for 2 g tube</td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<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>
		<tr height="21">
			<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>
		</tr>
		<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 imaged using static AFM imaging. As a control for the assembly and deposition, H3 mono-nucleosomes were prepared and imaged using static AFM. An image of the H3 mono-nucleosomes (Figure 4A) provides a snapshot of the nucleosome population as it existed moments before deposition, confirming that nucleosomes were successfully assembled. The 2 nM nucleosome deposition provided a uniform distribution of nucleosome and DNA particles across the surface and very little to no crowding was observed.
With the H3 control assembly a success, the presented methods were next applied to the study of CENP-A nucleosomes. Static AFM imaging of this sample (Figure 4B) revealed that the assembly was a success. To demonstrate the influence of nucleosome concentration on the surface particle density, the CENP-A nucleosomes were deposited at 1 nM (Figure 4B), compared to the 2 nM used for H3 (Figure 4A). This resulted in a reduced surface particle density for the CENP-A sample to approximately half that of H3 sample. From the static AFM images, the height and turn number of mono-nucleosomes were characterized (Figure 5). Both the angle between the free DNA arms and the length of the free DNA arms was used to determine the number of DNA turns in the individual nucleosome.
Time-lapse AFM imaging of the nucleosomes in buffer was used to visualize the overall spontaneous unwrapping behavior of the nucleosomes (Figure 6). Measuring the angle between nucleosome arms and the contour length of the arms allowed for the turn number to be determined in each of the frames during this unwrapping process (Figure 6 B-C). As the turn number of the nucleosome decreases, a corresponding decrease in the nucleosome core volume is also observed (Figure 6C). High-speed time-lapse AFM was next used to probe the more intricate nucleosome dynamics that were missed using standard time-lapse imaging. The ability of this technique to capture the dynamics over a long period of time was essential to the visualization of a long-distance translocation of a CENP-A nucleosome core (Figure 7) which was captured over the course of ~1200 frames. This technique was also critical in capturing the rare transfer of a CENP-A nucleosome core from one DNA substrate to another (Figure 8). The fast image capture rate (~300 ms/frame) made visualization of this dynamic event possible, as it only took several frames to complete.&#160; &#160;
Table 1: Reagents needed for continuous salt gradient nucleosome assembly. Each of the components listed is added to the microfuge tube containing the purified DNA. This should be done in the order in which the reagents are listed in the table, with water and NaCl added first, followed by the H2A/H2B dimer and the histone tetramer added last. If pre-folded histone octamers are to be used, add at the same ratio as for the tetramer above. *Take note of the NaCl content in each of the histone stocks and adjust the 5M NaCl to add accordingly, the final [NaCl] should equal 2M.&#160; (See Table of Materials).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58820/58820fig1.jpg" /> 
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.30 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58820/58820fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58820/58820fig2.jpg" /> 
Figure 2: SDS-PAGE of assembled nucleosomes. Lanes 1 and 2 contain the H3 octamer and the CENP-A assembly of histones, respectively. Lanes 3 and 4 contain the assembled H3 nucleosomes and the assembled CENP-A nucleosomes, respectively. Comparison of the assembled nucleosomes to the histone only controls in lanes, confirm that nucleosomes were properly assembled. The cartoon schematic above each lane indicates which histone components are present. This figure is adapted from Stumme-Diers et al.30 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58820/58820fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58820/58820fig3.jpg" /> 
Figure 3: Schematic of the process to prepare APS functionalized mica for AFM imaging of nucleosomes. (A) a piece of mica ~0.1 mm in thickness has both sides freshly cleaved. (B) The cleaved mica piece is promptly placed diagonally in a cuvette containing the APS solution and is set to incubate for 30 min. (C) Following the APS functionalization step, the APS-mica piece is transferred to a cuvette filled with dd H2O for a 30 s rinse. (D) The APS-mica piece is stored in a cuvette until use. &#160; <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58820/58820fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58820/58820fig4.jpg" /> 
Figure 4: Example AFM images of H3 and CNEP-A nucleosomes. (A) Sample image of H3 mono-nucleosomes deposited on APS-mica, captured using static AFM. Each bright blob is a nucleosome core particle with the flanking DNA regions appearing as noodle-like arms. The long noodle-like features are free DNA particles that are not associated with a histone core. For this image, a 2 nM nucleosome concentration was used, providing a uniform distribution across the surface, with little to no crowding. (B) This nucleosome sample was deposited at 1 nM and is much less populated than the 2 nM used in (A). This demonstrates the direct effect that nucleosome dilution has on the surface density of nucleosomes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58820/58820fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58820/58820fig5.jpg" /> 
Figure 5: Visual depiction of the analysis used to characterize the wrapping and height of the nucleosome particles. (A) A representative nucleosome particle from images like those shown in Figure 3. The contour length of each nucleosome arm is measured from the end of the arm to the center of the core (dotted green lines). &#160;Plotting cross section profiles (red and blue lines) of a nucleosome produce the curves shown in (B)&#160;From these curves, height and width detail of particle can be determined.&#160; (C) Schematic of the various wrapped states of the nucleosomes. (D) Each wrapped state is characterized using the angle between the DNA arms, the number of DNA turns and the bp of wrapped DNA. Scale Bar = 20 nm. This figure is adapted from Lyubchenko et al. 24 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58820/58820fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58820/58820fig6.jpg" /> 
Figure 6. Example of time-lapse AFM images capturing the spontaneous unwrapping of nucleosomes. (A) A series of consecutive AFM images of the spontaneous unwrapping process of nucleosomes captured by continuous scanning in the buffer. The size of each frame is 200 nm and images were captured at a rate of ~170 s per frame. (B) As the unwrapping process progresses in each frame, the arm lengths of the nucleosome increase, (C) resulting in a decrease in DNA turns around the nucleosome. This turn number can be determined from either the measured arm lengths (black) or the angle between the nucleosome arms (red). As the turn number decreases, a reduction in nucleosome volume is also observed (blue curve, right axis). Each frame is 200 x 200 nm in size. This figure is adapted from Lyubchenko et al. 24 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58820/58820fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58820/58820fig7.jpg" /> 
Figure 7. Demonstration of the high capacity of high-speed time-lapse AFM for long image acquisition times (A) A gallery of images selected from more than 1200 frames demonstrating the translocation behavior of a CENP-A nucleosomes core. Each image was captured at a rate of ~300 ms/frame. (B) Contour length measurements from one end of the DNA substrate to the CENP-A core are used to characterize this long translocation process. Scale Bar = 25 nm. This figure is adapted from Stumme-Diers et al. 16 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58820/58820fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58820/58820fig8.jpg" /> 
Figure 8. Example of a dynamic nucleosome core transfer captured using high-speed time-lapse AFM (figure adapted from Stumme-Diers et al.16&#160;(A) Selected frames demonstrating the spontaneous transfer of a CENP-A nucleosome core from one DNA substrate to another. This process took place within several frames which were captured at a rate of ~300 ms/frame. (B) A schematic of the transfer process shown in (A). Scale Bar = 25 nm. This figure is adapted from Stumme-Diers et al. 16 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58820/58820fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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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11.5K Views.  University of Nebraska Medical Center. 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.

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

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Investigating Protein Sequence-structure-dynamics Relationships with Bio3D-web

We demonstrate the usage of Bio3D-web for the interactive analysis of biomolecular structure data. The Bio3D-web application provides online functionality for: (1) The identification of related protein structure sets to user specified thresholds of similarity; (2) Their multiple alignment and structure superposition; (3) Sequence and structure conservation analysis; (4) Inter-conformer relationship mapping with principal component analysis, and (5) comparison of predicted internal dynamics via ensemble normal mode analysis. This integrated functionality provides a complete online workflow for investigating sequence-structure-dynamic relationships within protein families and superfamilies.

A protocol for the online investigation of protein sequence-structure-dynamics relationships using Bio3D-web is presented.

We demonstrate the usage of Bio3D-web for the interactive analysis of biomolecular structure data. The Bio3D-web application provides online functionality ...

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

Structure Solution of the Fluorescent Protein Cerulean Using MeshAndCollect

X-ray crystallography is the major technique used to obtain high resolution information concerning the 3-dimensional structures of biological macromolecules. Until recently, a major requirement has been the availability of relatively large, well diffracting crystals, which are often challenging to obtain. However, the advent of serial crystallography and a renaissance in multi-crystal data collection methods has meant that the availability of large crystals need no longer be a limiting factor. Here, we illustrate the use of the automated MeshAndCollect protocol, which first identifies the positions of many small crystals mounted on the same sample holder and then directs the collection from the crystals of a series of partial diffraction data sets for subsequent merging and use in structure determination. MeshAndCollect can be applied to any type of micro-crystals, even if weakly diffracting. As an example, we present here the use of the technique to solve the crystal structure of the Cyan Fluorescent Protein (CFP) Cerulean.

We present the use of the MeshAndCollect protocol to obtain a complete diffraction data set, for use in subsequent structure determination, composed of partial diffraction data sets collected from many small crystals of the fluorescent protein Cerulean.

X-ray crystallography is the major technique used to obtain high resolution information concerning the 3-dimensional structures of biological ...

Imaging of Extracellular Vesicles by Atomic Force Microscopy

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane proteins and other molecules, and diverse luminal content inherited from a parent cell, which includes RNAs, proteins, and DNAs. The characterization of the hydrodynamic sizes of EVs, which depends on the size of the vesicle and its coronal layer formed by surface decorations, has become routine. For exosomes, the smallest of EVs, the relative difference between the hydrodynamic and vesicles sizes is significant. The characterization of vesicles sizes by the cryogenic transmission electron microscopy (cryo-TEM) imaging, a gold standard technique, remains a challenge due to the cost of the instrument, the expertise required to perform the sample preparation, imaging and data analysis, and a small number of particles often observed in images. A widely available and accessible alternative is the atomic force microscopy (AFM), which can produce versatile data on three-dimensional geometry, size, and other biophysical properties of extracellular vesicles. The developed protocol guides the users in utilizing this analytical tool and outlines the workflow for the analysis of EVs by the AFM, which includes the sample preparation for imaging EVs in hydrated or desiccated form, the electrostatic immobilization of vesicles on a substrate, data acquisition, its analysis, and interpretation. The representative results demonstrate that the fixation of EVs on the modified mica surface is predictable, customizable, and allows the user to obtain sizing results for a large number of vesicles. The vesicle sizing based on the AFM data was found to be consistent with the cryo-TEM imaging.&#160;

A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties.&#160;

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with neurodegenerative conditions such as Alzheimer&#8217;s and Parkinson&#8217;s diseases. In particular low molecular weight aggregates, amyloid oligomers, have been shown to possess generic cytotoxic properties and are implicated as neurotoxins in many forms of dementia. We illustrate the use of methods based on atomic force microscopy (AFM) to address the challenging task of characterizing the morphological, structural and chemical properties of these aggregates, which are difficult to study using conventional structural methods or bulk biophysical methods because of their heterogeneity and transient nature. Scanning probe microscopy approaches are now capable of investigating the morphology of amyloid aggregates with sub-nanometer resolution. We show here that infrared (IR) nanospectroscopy (AFM-IR), which simultaneously exploits the high resolution of AFM and the chemical recognition power of IR spectroscopy, can go further and enable the characterization of the structural properties of individual protein aggregates, and thus offer insights into the aggregation mechanisms. Since the approach that we describe can be applied also to the investigations of the interactions of protein assemblies with small molecules and antibodies, it can deliver fundamental information to develop new therapeutic compounds to diagnose or treat neurodegenerative disorders.

We describe the application of infrared nanospectroscopy and high-resolution atomic force microscopy to visualize the process of protein self-assembly into oligomeric aggregates and amyloid fibrils, which is closely associated with the onset and development of a wide range of human neurodegenerative disorders.

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with ...

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and provides a theoretical basis for the rational design of nano-medicine delivery. Single particle tracking (SPT) analysis could probe the position and orientation of individual nanoparticles on cell membrane, and reveal their translational and rotational states. Here, we show how to use traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on live cell membrane. We also show how to extract the location and orientation of AuNRs using ImageJ and MATLAB, and how to characterize the diffusive states of AuNRs. Statistical analysis of hundreds of particles show that single AuNRs perform Brownian motion on the surface of U87 MG cell membrane. However, individual long trajectory analysis shows that AuNRs have two distinctly different types of motion states on the membrane, namely long-range transport and limited-area confinement. Our SPT methods can be potentially used to study the surface or intracellular particle diffusion in different biological cells and can become a powerful tool for investigations of complex cellular mechanisms.

Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis.

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and ...