This work is supported in part by the University of Mississippi Graduate Student Council Research Fellowship (OA), University of Mississippi Sally McDonnell-Barksdale Honors College (JCW, JER), the Mississippi Space Grant Consortium under grant number NNX15AH78H (JCW, DNR), and the American Heart Association under grant number 848586 (DNR).

1. Etching coverslips
<ol>
	<li>Dissolve 100 g of KOH in 300 mL of 100% ethanol in a 1,000 mL beaker. Stir with a stir bar until the majority of the KOH has dissolved. 
	CAUTION: Concentrated KOH solution can cause burns and damage to clothing. Wear gloves, eye protection, and a lab coat.</li>
	<li>Place coverslips individually in coverslip cleaning racks. 
	NOTE: Racks are designed with slits that hold single coverslips spaced apart to allow for etching and rinsing on each face of the covers...

The authors have no conflicts of interest to declare.

An in vitro study using optical tweezers combined with fluorescence imaging was performed to investigate the dynamics of myosin ensembles interacting with actin filaments. Actin-myosin-actin bundles were assembled using muscle myosin II, rhodamine actin at the bottom of the bundle and on the coverslip surface, and 488-labeled, biotinylated actin filaments on the top of the bundle. Actin protein from rabbit muscle was polymerized and stabilized using general actin buffers (GAB) and actin polymerizing buffers (APB...

Motor proteins are essential to life, converting chemical energy into mechanical work1,2,3. Myosin motors interact with actin filaments by taking steps along the filaments similar to a track, and the dynamics of actin-myosin networks carry out muscle contraction, cell motility, the contractile ring during cytokinesis, and movement of cargo inside the cell, among other essential tasks3,4,5,6,7,8. Since myosins have so many essential roles, failure in the functionality of the myosin-actin network can lead to disease development, such as mutations in the myosin heavy chain that cause heart hypercontractility in hypertrophic cardiomyopathy (HCM)9,10,11,12,13,14. In muscle contraction, individual myosin motors cooperate with each other by working as an ensemble to provide the required mechanical energy that carries out the relative sliding of AFs4,15,16,17,18. Myosin motors form crossbridges between AFs and use conformational changes due to its mechanochemical cycle to collectively move toward the barbed end of the aligned filaments17,18,19,20,21.
Development of quantitative in vitro motility assays at the SM level using techniques such as optical trapping has facilitated gathering unprecedented detail of how individual myosin motors function, including measuring SM force generation and step sizes22,23,24,25,26,27,28,29,30. Finer et al. developed the &#34;three-bead&#34; or &#34;dumbbell&#34; optical trapping assay to probe the force-generation mechanics of single myosin II motors23,31. As muscle myosin II works in teams to contract AFs but is non-processive at the SM level, the optical trapping assay orientation had to be rearranged from the classic motor-bound bead approach32. To form the dumbbell assay, two optical traps were used to hold an AF over a myosin motor bound to a coverslip-attached bead, and force output by the single motor was measured through movements of the AF within the trap23.
However, SM forces and using a single motor/single filament assay orientation do not give a full image about system-level force generation since many motor proteins, including myosin II, do not work in isolation and often do not function as a sum of their parts15,16,17,32,33,34,35,36. More complex structures that include more than one motor interacting with more than one filament are necessary to better understand the synergy of myosin and actin filaments&#39; networks15,32. The dumbbell assay orientation has been exploited to investigate small ensemble force generation by having multiple myosins attached to a bead or using a myosin-thick filament attached to a surface and allowing the motors to interact with the suspended AF4,23,34,37,38,39,40.
Other small ensemble assays include an in vitro filament gliding assay wherein myosin motors are coated onto a coverslip surface, and a bead bound to an AF is used to probe the force generated by the team of motors4,35,36,38,39,40,41,42,43. In both these cases, the myosins are bound to a rigid surface &#8211; bead or coverslip &#8211; and utilize one AF. In these cases, the motors are not able to move freely or communicate with each other, nor does having myosins rigidly bound reflect the compliant, hierarchical environment in which the motors would work together in the sarcomere32. Previous studies have suggested that myosin II can sense its environment and adapt accordingly to changing viscoelastic or motor concentration conditions by altering characteristics such as force generation and duty ratio41,44,45. Thus, there is a need to develop an optical trapping assay that fosters and captures motor communication and system compliancy to paint a more realistic picture of the mechanistic underpinnings of myosin II ensemble force generation.
Here, we developed a method to couple hierarchical structure in vitro with optical trapping by forming actomyosin bundles or sandwiches consisting of multiple myosin motors interacting between two actin filaments. This modular assay geometry has the ability to directly probe how molecular and environmental factors influence ensemble myosin force generation. Further, investigating force generation mechanisms through these actin-myosin ensembles have the potential to aid in modeling and understanding how large-scale cellular tasks, such as muscle contraction, propagate up from the molecular level9,10,13.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Actin protein (biotin): skeletal muscle</td>
			<td>Cytoskeleton</td>
			<td>AB07-A</td>
			<td>Biotinylated actin protein</td>
		</tr>
		<tr>
			<td>Actin protein, rabbit skeletal muscle</td>
			<td>Cytoskeleton</td>
			<td>AKL99-A</td>
			<td>Actin protein</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Phalloidin</td>
			<td>Invitrogen</td>
			<td>A12379</td>
			<td>Actin stabilizer and Alexa Fluor 488 stain</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Fisher scientific</td>
			<td>BP413-25</td>
			<td>Required for actin assembly and myosin motility</td>
		</tr>
		<tr>
			<td>Beta-D-glucose</td>
			<td>Fisher scientific</td>
			<td>MP218069110</td>
			<td>Part of oxygen scavenging system used to reduce photobleaching during fluorescence imaging</td>
		</tr>
		<tr>
			<td>Blotting Grade Blocker (casein)</td>
			<td>Biorad</td>
			<td>1706404</td>
			<td>Used to block surface from non-specific binding</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Fisher scientific</td>
			<td>C79500</td>
			<td>Calcium chloride, provides the necessary control over the dynamics of actin myosin network</td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Fisher scientific</td>
			<td>ICN10040280</td>
			<td>Part of oxygen scavenging system used to reduce photobleaching during fluorescence imaging</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Fisher scientific</td>
			<td>12544C</td>
			<td>Used to make flow cells</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Fisher scientific</td>
			<td>AC327190010</td>
			<td>Used for buffer preparation</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher scientific</td>
			<td>A4094</td>
			<td>Regent used for cleaning coverslips</td>
		</tr>
		<tr>
			<td>Glucose oxidase</td>
			<td>Fisher scientific</td>
			<td>34-538-610KU</td>
			<td>Part of oxygen scavenging system used to reduce photobleaching during fluorescence imaging</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher scientific</td>
			<td>P217-500</td>
			<td>Used for buffer preparation</td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Fisher scientific</td>
			<td>P250-1</td>
			<td>Used to etch coverslips and adjust buffer pH</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Fisher scientific</td>
			<td>M33-500</td>
			<td>Used for buffer preparation</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Fisher scientific</td>
			<td>12-544-2</td>
			<td>Used to make flow cells</td>
		</tr>
		<tr>
			<td>Myosin II protein: rabbit skeletal muscle</td>
			<td>Cytoskeleton</td>
			<td>MY02</td>
			<td>Full length myosin motor protein isolated from rabbit skeletal muscle</td>
		</tr>
		<tr>
			<td>Nanotracker2</td>
			<td>Bruker/JPK</td>
			<td>NT2</td>
			<td>Optical trapping instrument</td>
		</tr>
		<tr>
			<td>Poly-l-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P8920</td>
			<td>Facilities adhesion of actin filaments onto glass surface of the coverslip</td>
		</tr>
		<tr>
			<td>Rhodamine Phalloidin</td>
			<td>Cytoskeleton</td>
			<td>PHDR1</td>
			<td>Actin stabilizer and rhodamine fluorescent stain</td>
		</tr>
		<tr>
			<td>Streptavidin beads, 1 &#956;m</td>
			<td>Spherotech</td>
			<td>SVP-10-5</td>
			<td>Optical trapping handle</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Fisher scientific</td>
			<td>PR H5121</td>
			<td>Used for buffer preparation</td>
		</tr>
	</tbody>
</table>

probing myosin ensemble mechanics in actin filament bundles using optical tweezers

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle contraction. To understand their force-generating mechanisms, myosin II has been investigated both at the single-molecule (SM) level and as teams of motors in vitro using biophysical methods such as optical trapping.
These studies showed that myosin force-generating behavior can differ greatly when moving from the single-molecule level in a three-bead arrangement to groups of motors working together on a rigid bead or coverslip surface in a gliding arrangement. However, these assay constructions do not permit evaluating the group dynamics of myosin within viscoelastic structural hierarchy as they would within a cell. We have developed a method using optical tweezers to investigate the mechanics of force generation by myosin ensembles interacting with multiple actin filaments.
These actomyosin bundles facilitate investigation in a hierarchical and compliant environment that captures motor communication and ensemble force output. The customizable nature of the assay allows for altering experimental conditions to understand how modifications to the myosin ensemble, actin filament bundle, or the surrounding environment result in differing force outputs.

Flow cells containing the actomyosin bundle systems are of a standard design, consisting of a microscope slide and an etched coverslip separated by a channel made from double-sided sticky tape (Figure 1). The assay is then built from the coverslip up using staged introductions as described in the protocol. The final assay consists of template rhodamine-labeled actin filaments; the desired myosin concentration (1 &#956;M was used for the representative results in Figure 2...

Watch this Scientific Journal Video about Probing Myosin Ensemble Mechanics in Actin Filament Bundles Using Optical Tweezers at JoVE.com

Probing Myosin Ensemble Mechanics in Actin Filament Bundles Using Optical Tweezers

Formation of actomyosin bundles in vitro and measuring myosin ensemble force generation using optical tweezers is presented and discussed.

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle ...

Investigating actin behavior using this protocol is significant because it allows the user to probe motor protein ensemble dynamics using the precision of optical trapping. The main advantage of this technique is the ability to measure myosin mechanics within actin structural hierarchy, as opposed to the traditional single-motor single-filament orientation used in many optical trapping experiments. Another advantage of this protocol is its modular nature, which allows the user to customize the assay to the hierarchical cytoskeleton system of their choosing.To begin, apply two pieces of double-sided sticky tape to the middle of a microscope slide three to four millimeters apart from each other. Then tear or cut off the excess tape that hangs off the edge of the slide. Next, add the PLL-coated cover slip on top of the tape perpendicular to the long axis of the microscope slide to form a channel.Using a small tube to compress the cover slip onto the tape and microscope, slide thoroughly until the tape is transparent, ensuring that there are no bubbles in the tape as this can cause leakage from the flow channel. Add 15 microliters of the diluted rhodamine actin to the PLL flow cell and wick the excess solution through the flow cell, but do not allow the flow channel to become dry. Then, incubate for 10 minutes in a humidity chamber.Next, prepare a one milligram per milliliter casein solution in actin polymerization buffer and add 15 microliters of the solution to prevent non-specific binding of the subsequent components. Then, incubate for five minutes in a humidity chamber. Afterward, add the desired concentration of myosin to the prepared biotinylated actin and bead suspension and gently stir with a pipette tip.Then, immediately add 15 microliters of the suspension along with the desired myosin concentration to the flow cell and incubate for 20 minutes. Next, seal the open ends of the flow cell with nail polish to prevent evaporation during imaging and optical trapping experiments. Once the system is ready, turn on the laser using the laser power button at the left bottom corner of the screen to 50 milliwatts and let it stabilize for 30 minutes.Sequentially click on the illumination, camera, objective, and stage movement buttons within the software to bring up those windows for viewing and manipulation during the experiment. Turn the microscope illumination on by clicking on the On/Off button and setting it to maximum power by clicking and dragging the bar all the way to the right. Open the sample area and remove the sample holder from the microscope stage.Then add the flow cell and secure it with the metal sample holders, ensuring that the cover slip is on the bottom. Afterward, add 30 microliters of RO water to the center of the bottom objective and reinsert the sample stage. Then raise the lower objective using the L2 on the remote controller until the bead of water touches the cover slip.Next, lower the top objective until about half the distance to the flow cell is reached using the R2 on the remote controller. Then, add 170 microliters of RO water to the top of the flow cell directly under the top objective and lower the top objective until it breaks the surface tension of the water and forms a meniscus. Subsequently, move the microscope stage using the arrow pad on the remote controller until the edge of the tape adjacent to the flow channel is reached and then close the sample door.Using the objective window in the screen, bring the edge of the tape and focus by bringing the bottom objective named Laser Objective up by clicking on the upper arrow and the top objective by clicking bottom arrow using the onscreen controls. Find a floating bead and trap it by clicking on the trap shutter button, which will open the shutter and allow the trapping laser to hit the sample. Then, click on the trap cursor on the screen and drag it to move the location of the trapping laser.Once trapped, calibrate the bead by clicking on the calibration button. Then, click on Settings and type in the diameter of the bead and temperature of the stage found in the bottom left of the software window. Next, click on Trap 1, then on X Signal and perform the corner frequency fit by clicking on Run.Then click and drag within the window to optimize the function fit. Afterward, click on Use it for sensitivity and stiffness values and then accept values. Following, close the window.Find an actomyosin bundle by searching for beads bound to AFs on the surface of the cover slip and by looking for both fluorescent AFs co-localized. Turn on the white light source and use the appropriate filter cube to image each actin filament by turning the turret. Once verified, trap the bead attached to the top filament of the bundle by clicking on the trap shutter button.Then, record the data by clicking on the oscilloscope button using the onscreen controls. To visualize measurements without recording the data, click on Start and then click on Autosave to save all the data. To record measurements, click on Start record and choose which data are to be visualized in real time by choosing from the dropdown menu X Signal or Y Signal.The force trace of skeletal myosin motors generating force within the constructed in vitro actin structural hierarchy exhibit steady ramp in force until a plateau is reached over two to five minutes. However, it is also observed that some actomyosin bundles that do not generate any net force and the force traces appear as baseline noise or exhibit no substantial net increase in force over 90 seconds due to a low concentration of motor or an unfavorable parallel orientation of the filaments. This protocol development will pave the way toward building more complex in vitro cytoskeletal assays that will further help model large-scale cellular tasks, such as cell division and muscle contraction from the bottom up.

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2.0K 조회수.  University of Mississippi. 이 프로토콜을 사용하여 액틴 거동을 조사하는 것은 사용자가 광학 트래핑의 정밀도를 사용하여 모터 단백질 앙상블 역학을 조사할 수 있기 때문에 중요합니다. 이 기술의 주요 장점은 많은 광학 트래핑 실험에 사용되는 기존의 단일 모터 단일 필라멘트 방향과 달리 액틴 구조 계층 구조 내에서 미오신 역학을 측정 할 수 있다는 것입니다. 이 프로토콜의 또 다른 장점은 사용...