eoe sub articles

EoE Sub Articles

1. Gliding actin filament assay
<ol>
	<li>Coverslip preparation
	<ol>
		<li>Make a 1% nitrocellulose solution in amyl acetate.</li>
		<li>Obtain a tissue culture dish (150 x 25 mm) and add a circular filter paper (125 mm diameter) to the bottom of the dish.</li>
		<li>Load eight No. 1.5 thickness 22 mm square coverslips onto a rack and wash with approximately 2-5 mL of 200-proof ethanol followed by 2-5 mL of distilled water (dH2O). Repeat this washing step, ending with water. Then, dry the c...

the gliding actin filament assay: an in vitro motility assay to study translocation of actin filaments on immobilized myosins

Source: Tripathi, A., et al. <a href="https://app.jove.com/v/62180">Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays.</a> J. Vis. Exp. (2021).
In this video, we perform an in vitro motility assay to analyze the actin-myosin interactions. The gliding movement of actin filaments on immobilized myosin molecules is visualized using fluorescence microscopy.

<img alt="Figure 1" src="/files/ftp_upload/21215/21215_Figure1.jpg" /> 
Figure 1: Preparation of functionalized flow-cell chambers. (A) Begin with a cleaned microscope slide, two pieces of double-sided tape cut to approximately 2 cm, and a functionalized coverslip. (B) Add the tape to the center of the microscope slide. (C) Attach the coverslip to the tape with the coating (i.e., nitrocellulose) facing down and gentl...

The Gliding Actin Filament Assay: An In Vitro Motility Assay to Study Translocation of Actin Filaments on Immobilized Myosins

Source: Tripathi, A., et al. Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays. J. Vis. Exp. (2021).
In this video, we ...

Myosin - a motor protein - interacts with another filamentous protein, actin, causing its translocation.
To analyze the gliding movement of actin, assemble a glass slide-based flow chamber such that the inner portion of the chamber-top is nitrocellulose-coated. Flow in the myosin proteins through the chamber, which immobilize over the nitrocellulose coating.
Supplement the chamber with unlabeled actin filaments - black actins - and add a motility buffer containing adenosine triphosphate or ATP molecules.
The actin filaments bind to immobilized active myosins along with inactive or dead myosins. ATP molecules interact with the active myosins, leading to their dissociation from black actins while the inactive myosins remain actin-bound.
Centrifuge to pelletize inactive myosins attached to black actins. Rinse with a buffer thoroughly to remove the inactive myosin pellet, free actin filaments, and unused ATPs. Flow in fluorescently-labeled actins that bind immobilized, active myosins.
Supplement the chamber with a final buffer containing a crowding agent - methylcellulose, and ATP. The crowding agent forces actin filaments to remain closer to the chamber surface.
Each active myosin hydrolyzes ATP leading to conformational change and binding to a new position along the filament, generating a power stroke that glides the actin over myosin.
Observe the chamber under a fluorescence microscope to record the motion of labeled actin filaments gliding smoothly over the immobilized myosin heads, confirming successful translocation.

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Research

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Biochemical assays

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

JoVE Journal

Biochemistry

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.

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 ...

Bioengineering

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...

The Gliding Actin Filament Assay: An In Vitro Motility Assay to Study Translocation of Actin Filaments on Immobilized Myosins

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