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>
			<td style="width:327px;">Addgene, Cambridge, MA</td>
			<td style="width:119px;">26656</td>
			<td style="width:449px;"></td>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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			<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>
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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 ...

This protocol utilizes all major features of AFM, non-amenable to other single molecule techniques. As a result, we are able to resolve structure of nucleosome at nano scale. Importantly, direct visualization of nucleosomes was done at physiological conditions.Using our AFM technique, we have recently revealed unique properties of the centromere nucleosome that can play a role on the function of the entire centromere. The method to be shown is currently applied in our lab to investigate self-assembly of proteins associated with formation of amyloid aggregates. These protein aggregates trigger the development of devastating diseases such as Alzheimer's and Parkinson's to name a few.The simple preparation methods are also applicable to other protein-DNA complexes, including large systems such as DNA replication machinery. Although some modifications are needed. In order to characterize nucleosome particles at the single molecule level using static and time-lapse atomic force microscopy, begin by purifying, assembling, and validating the nucleosomes as described in the accompanying text protocol.Next, prepare the mica surface for static AFM imaging. To do this, first prepare a fifty millimolar APS stock solution in deionized water. Store one milliliter aliquots of this solution at four degrees Celsius, until it is needed.From the stock solution, prepare a working APS solution for mica modification by dissolving 50 microliters of the 50 micromolar APS stock in 15 milliliters of distilled deionized water. Mix the solution and then fill a cuvette with the solution. Next, cut one by three centimeter strips of mica from high quality mica sheets.Check that the piece fits when placed diagonally in a cuvette. Then, cleave layers of the mica until both sides are freshly cleaved, and the piece is as thin as 0.1 millimeter. Immediately place the mica piece into the APS filled cuvette and incubate the mica for 30 minutes.Transfer the mica piece to a cuvette filled with distilled deionized water and soak it for 30 seconds. Then, use argon to completely dry both sides of the APS mica strip. Apply double faced adhesive tape to several magnetic pucks and place them to the side.Then, cut the APS mica substrate to one centimeter by one centimeter squares, and cover them in a clean Petri dish. Next, prepare three dilutions of the assembled nucleosomes using a 0.22 micron filtered buffer containing 10 millimolar hepes and four millimolar magnesium chloride at a PH of 7.5. To limit the loss of nucleosomes, at the low final concentration, the dilution should be done one at a time, immediately prior to deposition on the APS mica.Deposit five to 10 microliters of each nucleosome sample at the center of an APS mica piece and let them incubate for two minutes. Then, gently rinse the sample with two to three milliliters of distilled deionized water to remove the buffer components. And dry the deposited sample under a light flow of argon.To begin, mount a tip on the tip holder of the AFM setup. Then, mount the first sample on the AFM stage, being careful not to contact the sample surface. Position the laser over the cantilever until its sum is at the maximum and adjust the vertical and lateral deflection values to near zero.Then, tune the AFM probe to find its resonant frequency, adjust the drive amplitude, and set the image size to 100 by 100 nanometers. Once set up, click the engage button to begin the approach. When the approach is complete, gradually optimize the amplitude set point until the surface of the sample is clearly seen.Then, increase the scan size to one micron by one micron and the resolution to 512 by 512 pixels. Finally, click the capture button to begin image acquisition. Use glass rod scanner glue to attach a glass rod to the AFM scanner stage.Allow this to dry for a minimum of 10 minutes. In the meantime, make 0.1 millimeter thick circular pieces of mica with a 1.5 millimeter diameter by punching them from a larger mica sheet. Use the high speed AFM mica glass rod glue to attach this mica piece to the glass rod on the high speed AFM, and allow it to remain untouched for a minimum of 10 minutes while it dries.Then, cleave layers from the mica using a pressure sensitive tape until a well cleaved layer is seen on the tape. Next, dilute one microliter of 50 millimolar APS stock in 99 microliters of distilled deionized water to make a 500 micromolar APS solution. Deposit 2.5 microliters of the solution on the freshly cleaved mica surface and let the surface functionalize for 30 minutes.Following the incubation, rinse the mica several times with distilled deionized water by applying three microliters rinses. Remove the water completely following each rinse by placing a non-woven wipe at the edge of the mica. After the final rinse, place three microliters of distilled deionized water on the surface, and let it sit for a minimum of five minutes to remove any non-specifically bound APS.Next, place the probe in the high speed AFM holder, and position the holder on the AFM stage with the tip facing up. Rinse the holder using 100 microliters of distilled deionized water, followed by two 100 microliter rinses of 0.22 micron filtered nucleosome imaging buffer. With the rinses done, fill the chamber with 100 microliters of nucleosome imaging buffer, submerging the tip.Adjust the cantilever position until it is hit with the laser. Then, rinse the APS mica five times with filtered nucleosome imaging buffer, using four microliters per rinse. Dilute one microliter of the nucleosome assembly stock into 250 microliters of filtered nucleosome imaging buffer for a final nucleosome concentration of one nanomolar.Deposit 2.5 microliters of this dilution on the surface and let it sit for two minutes. Rinse the surface twice with four microliters of nucleosome imaging buffer to avoid over crowding. After the final rinse, leave the surface covered in imaging buffer.Set the scanner and sample on top of the tip holder so that the sample is face down. To begin the approach, use the auto approach function with the set point amplitude close to the free oscillation amplitude. Adjust the set point until the surface is being well tracked.Then, set the image area around 150 by 150 nanometers to 200 by 200 nanometers with the data acquisition rate of around 300 milliseconds per imaging frame. As a control for the assembly and deposition, H3 mononucleosomes were prepared and imaged using static AFM. This image provides a snapshot of the nucleosome population as it existed moments before deposition confirming that nucelosomes were successfully assembled.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 revealed its assembly was a success. From the static AFM images, the height and turn number of mononucleosomes could be characterized.Both the angle between the free DNA arms and the length of the free DNA arms are used to determine the number of DNA turns in the individual nucleosome. In contrast, time lapse AFM imaging of the nucleosomes can be used to visualize the overall spontaneous unwrapping behavior of the nucleosomes. As the turn number of the nucleosome decreases, a corresponding decrease in the nucleosome core volume is also observed.In addition to regular time lapse imaging, high speed time lapse AFM can be used to probe the more intricate nucleosome dynamics that is 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, 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.The fast image capture rate made visualization of this dynamic event possible as it only took several frames to complete. In the broad chromatin field there are a set of important questions relating to the roles of liger histones and histone magnification of the chromatin dynamics. All these questions can be answered using our technique.

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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.

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

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 ...

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 ...

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked&#160;bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...

Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor individual reactions live in well-controlled conditions. To do so, live single-filament experiments have emerged over the past 20 years, mostly using total internal reflection fluorescence (TIRF) microscopy, and have provided a trove of key results. In 2011, in order to further expand the possibilities of these experiments and to avoid recurring problematic artifacts, we introduced simple microfluidics in these assays. This study details our basic protocol, where individual actin filaments are anchored by one end to the passivated coverslip surface, align with the flow, and can be successively exposed to different protein solutions. We also present the protocols for specific applications and explain how controlled mechanical forces can be applied, thanks to the viscous drag of the flowing solution. We highlight the technical caveats of these experiments and briefly present possible developments based on this technique. These protocols and explanations, along with today&#39;s availability of easy-to-use microfluidics equipment, should allow non-specialists to implement this assay in their labs.

We present protocols for simple actin filament microfluidic assays, in combination with fluorescence microscopy, that allow one to accurately monitor individual actin filaments in real-time while sequentially exposing them to different protein solutions.

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor ...