Name: Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation (Video) | JoVE Journal
Uploaded: 2019-10-15T15:00:00.000+00:00
Duration: 8 min 9 s
Description: Learn to produce dynein and kinesin motor ensembles on DNA origami nanostructures for single molecule observation. This protocol enables precise control over motor placement for studying cooperative transport mechanisms.

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We thank K. Chau, J. Morgan, and A. Driller-Colangelo for contributing to the techniques of the segmented DNA origami chassis. We also thank former members of the Reck-Peterson and Shih laboratories for helpful discussions and contributions to the original development of these techniques. We thank J. Wopereis and the Smith College Center for Microscopy and Imaging and L. Bierwert and the Smith College Center for Molecular Biology. We gratefully acknowledge the NSF MRI program for the acquisition of a TIRF microscope.

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<li>Gennerich, A., Reck-Peterson, S. L. Chapter 4, Probing the force generation and stepping behavior of cytoplasmic Dynein. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGennerich%2C+A+%22Methods+in+Molecular+Biology%22">Methods in Molecular Biology</a>. , 63-80 (2011).</li>
<li>Goodman, B. S., Reck-Peterson, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+defined+motor+ensembles+with+DNA+origami%5BTitle%5D)+AND+%22Methods+in+Enzymology%22%5BJournal%5D)">Engineering defined motor ensembles with DNA origami.</a> Methods in Enzymology. 540, 169-188 (2014).</li>
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<li>Gietz, R. D. Yeast Transformation by the LiAc/SS Carrier DNA/PEG Method. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGietz%2C+RD+%22Yeast+Genetics.+Methods+in+Molecular+Biology+%28Methods+and+Protocols%29%22">Yeast Genetics. Methods in Molecular Biology (Methods and Protocols)</a>. 1205, Humana Press. New York, NY. (2014).</li>
<li>Hariadi, R. F., Cale, M., Sivaramakrishnan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myosin+lever+arm+directs+collective+motion+on+cellular+actin+network%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Myosin lever arm directs collective motion on cellular actin network.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (11), 4091-4096 (2014).</li>
<li>Krementsova, E. B., Furuta, K., Oiwa, K., Trybus, K. M., Ali, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small+teams+of+myosin+Vc+motors+coordinate+their+stepping+for+efficient+cargo+transport+on+actin+bundles%5BTitle%5D)+AND+%22The+Journal+of+Biological+Chemistry%22%5BJournal%5D)">Small teams of myosin Vc motors coordinate their stepping for efficient cargo transport on actin bundles.</a> The Journal of Biological Chemistry. 292 (26), 10998-11008 (2017).</li>
<li>Hariadi, R. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanical+coordination+in+motor+ensembles+revealed+using+engineered+artificial+myosin+filaments%5BTitle%5D)+AND+%22Nature+Nanotechnology%22%5BJournal%5D)">Mechanical coordination in motor ensembles revealed using engineered artificial myosin filaments.</a> Nature Nanotechnology. 10 (8), 696-700 (2015).</li>
<li>Hu, J. J., Morgan, J., Yang, Y., Lin, C., Derr, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spherical+DNA+Origami+as+a+Programmable+Cargo+Structure+for+Investigating+the+Emergent+Motility+of+Dynein+and+Kinesin+Ensembles%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">Spherical DNA Origami as a Programmable Cargo Structure for Investigating the Emergent Motility of Dynein and Kinesin Ensembles.</a> Biophysical Journal. 116 (3), 408A(2019).</li>
<li>Wagenbauer, K. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+We+Make+DNA+Origami%5BTitle%5D)+AND+%22Chembiochem%22%5BJournal%5D)">How We Make DNA Origami.</a> Chembiochem. 18 (19), 1873-1885 (2017).</li>
<li>Lin, C., Perrault, S. D., Kwak, M., Graf, F., Shih, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+of+DNA-origami+nanostructures+by+rate-zonal+centrifugation%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">Purification of DNA-origami nanostructures by rate-zonal centrifugation.</a> Nucleic Acids Research. 41 (2), e40(2013).</li>
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<li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NIH+Image+to+ImageJ%3A+25+years+of+image+analysis%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li>
<li>Reck-Peterson, S. L., Derr, N. D., Stuurman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+single+molecules+using+total+internal+reflection+fluorescence+microscopy+%28TIRFM%29%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Protocols%22%5BJournal%5D)">Imaging single molecules using total internal reflection fluorescence microscopy (TIRFM).</a> Cold Spring Harbor Protocols. 2010 (3), (2010).</li>
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</ol>

The authors have nothing to disclose.

The molecular construction techniques of DNA origami provide a unique way to construct motor ensembles with defined architectures, motor numbers, and types, enabling studies of how emergent behavior arises from specific motor configurations31. As structural and cellular studies continue to elucidate examples of cytoskeletal motors working in teams, techniques for isolating and investigating the biophysical and biochemical mechanisms of motors in ensembles are growing in utility. For example, cryo-...

Dynein and kinesin are cytoskeletal motor proteins responsible for myriad functions in eukaryotic cells1. By converting the chemical energy of ATP hydrolysis into productive work, these motors translocate on microtubules to haul and distribute various intracellular cargos. They also coordinate in the massive intracellular rearrangements associated with mitosis, where they exhibit orchestrated forces that contribute to the positioning and separation of chromosomes. Structural, biochemical, and biophysical assays, including single molecule observations, have revealed the mechanisms of these motors at the individual level (well-reviewed in previous works2,3,4). However, many of the motors&#39; tasks require them to work in small ensembles of both similar and mixed motor types. Comparatively less is understood about the mechanisms that coordinate the activity and ultimate emergent motility of these ensembles5,6. This knowledge gap is due, in part, to the difficulty in creating ensembles with controllable features, such as motor type and copy number. Over the past decade, the molecular construction techniques of DNA origami have been employed to solve this problem. For the microtubule based motors, some examples of these investigations include single molecule observations of ensembles of cytoplasmic dynein-17,8,9, intraflagellar dynein11, various kinesin motors12,13, and mixtures of both dyneins and kinesins7,14,15. Here, we provide details of the purification and oligonucleotide labeling of motors from yeast7,16,17,18,19,20, the folding and purification of segmented DNA origami with tunable compliance8, and the imaging of the yeast motors propelling the chassis structures7,18.
Constructing motor ensembles for in vitro single molecule observation requires three primary efforts. The first is the expression, purification and labeling of motor constructs suitable for attaching to DNA origami. The second is the production and purification of defined DNA origami structures (often termed &#34;chassis&#34;). And the third is the conjugation of the motors to the chassis structure followed by observation using total internal reflection fluorescence (TIRF) microscopy. Here, we provide established protocols for this process for recombinant microtubule-based motors purified from the yeast Saccharomyces cerevisiae7,16,17,18,19. DNA origami-based motor ensembles have been investigated using both recombinant kinesin15 and dynein7,8,18 constructs produced in this yeast expression system16,17,18,19. This protocol is valid for these constructs, given that they are controlled by the galactose induced promoter, and fused to the same protein tags for purification (ZZ and TEV protease linker) and for DNA oligo conjugation (SNAPtag).
Specific yeast strains produce specific motor constructs. For example, the dynein used to study the role of cargo compliance was purified from strain RPY10847,8. In general, strains containing motor constructs with the appropriate genetic modifications for expression and purification can be requested from the laboratories having published the use of those motors. Constructs with novel attributes such as mutations or tags can be made using recombinant genetic techniques, such as lithium acetate transformation21 and commercial kits. Detailed protocols for creating modified motor proteins in yeast for single molecule studies have been published19. In addition to the motors being fused to the SNAPtag, the oligos used to label the motors must be conjugated to the SNAP substrate, benzylguanine (BG); previously published protocols describe the formation and purification of BG-oligo conjugates18. The overall strategy described here has also been employed for actin-based motors (see previous works for examples22,23,24), and motors purified from other organisms and expression systems (see previous works for examples7,9,10,11,12,13,14).
Polymerized microtubules (MTs) are used in these experiments in two different procedures. MT affinity purification of functional motors requires MTs that are not labelled with other functional groups, while the motor-ensemble motility TIRF assay requires MTs labeled with biotin and fluorophores. In all cases, MTs are stabilized with taxol to prevent denaturation. The MT affinity purification step is used to remove any non-motile motors with a high MT affinity, as these motors can alter ensemble motility if conjugated to a chassis. During this process, active motors unbind the MTs and remain in solution, while tight-binding motors spin down in the MT pellet. This helps ensure all motors on the chassis are from an active population.
A variety of DNA origami structures have been used to study cytoskeletal motor ensembles. As the mechanistic understanding of ensemble transport has increased, the DNA origami structures employed in experiments have grown in complexity. In principle, any structure could be adapted for this purpose provided it is modified to include single-stranded DNA attachment sites for motors and fluorophores. Specific chassis designs and attributes may be useful for probing particular questions about the emergent behavior of motors ensembles. For example, rigid rods have been used to develop foundational knowledge of how copy number affects transport by teams of dyneins and kinesins7,15,18, and 2D platforms have been used to study myosin ensemble navigation of actin networks22. Structures with variable or tunable flexibility have been used to understand the roles of elastic coupling between motors and to probe how stepping synchronization affects motility8,24. More recently, spherical structures are being used to gain insight into how geometrical constraints to motor-track binding affect the dynamics of motility25.
In this protocol, we offer specific steps for ensemble experiments on segmented chassis with variable rigidity. Binding sites on the chassis are sometimes referred to as &#34;handles&#34;, while complementary DNA sequences that bind these handles are termed &#34;antihandles&#34;. The number of motors on these chassis is determined by which segments contain extended handle staples with complementarity to the antihandle oligo on the oligo-labeled motors. Using different handle sequences on different segments allows for binding of different types of motors to specific locations on the chassis. The chassis detailed here is composed of 7 sequential rigid segments, each comprised of 12 double-stranded DNA helices arranged in 2 concentric rings8. The rigid segments contain the motor handles and are connected through regions that can be either flexible single-stranded DNA or rigid double-stranded DNA, depending on the absence or presence, respectively, of &#34;linker&#34; staples. The compliance of the chassis structure is thus determined by the presence or absence of these &#34;linker&#34; staples. See previous reports for further details and specific DNA sequences8. In addition, multiple methods can be used to purify chassis26. The rate-zonal glycerol gradient centrifugation method27 is described here.

<table><tbody><tr><td>2 mL Round Bottom Tube</td><td>USA Scientific</td><td>1620-2700</td><td></td></tr><tr><td>Biotin labeled tubulin protein: porcine brain, &gt;99% pure</td><td>Cytoskeleton.com</td><td>T333P-A</td><td></td></tr><tr><td>Biotin-BSA</td><td>Sigma</td><td>A8549-10MG</td><td></td></tr><tr><td>Bottle Assembly, Polycarbonate, 250 mL, 62 x 120 mm</td><td>Beckman Coulter</td><td>356013</td><td></td></tr><tr><td>Bottle, with Cap Assembly, Polycarbonate, 10.4 mL, 16 x 76 mm</td><td>Beckman Coulter</td><td>355603</td><td></td></tr><tr><td>Centrifugal Filter Unit</td><td>Millipore Sigma</td><td>UFC30VV00</td><td></td></tr><tr><td>IgG Sepharose 6 Fast Flow, 10 mL</td><td>GE Healthcare</td><td>17096901</td><td></td></tr><tr><td>Micro Bio-Spin Chromatography Columns, empty</td><td>Bio-Rad</td><td>7326204EDU</td><td></td></tr><tr><td>P8064 Scaffold</td><td>Tilibit</td><td>2 mL at 400nM</td><td></td></tr><tr><td>Poly-Prep Chromatography Columns</td><td>Bio-Rad</td><td>731-1550</td><td></td></tr><tr><td>ProTev Protease</td><td>Promega</td><td>V6101</td><td></td></tr><tr><td>Scotch Double Sided Tape with Dispenser</td><td>amazon.com</td><td>N/A</td><td></td></tr><tr><td>Sephacryl S-500 HR</td><td>GE Healthcare</td><td>17061310</td><td></td></tr><tr><td>Streptavidin</td><td>Thermo Fisher</td><td>434302</td><td></td></tr><tr><td>SYBR Safe DNA stain</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Tubulin protein (&gt;99% pure): porcine brain</td><td>Cytoskeleton.com</td><td>T240-B</td><td></td></tr><tr><td>Tubulin, HiLyte 647</td><td>Cytoskeleton.com</td><td>TL670M-A</td><td></td></tr><tr><td>Ultra-Clear Centrifuge Tubes</td><td>Beckman Coulter</td><td>344090</td><td></td></tr></tbody></table>

production of dynein and kinesin motor ensembles on dna origami nanostructures for single molecule observation

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.

Successful purifications of motors and chassis structures were assayed by gel electrophoresis. SDS-PAGE analysis confirmed the successful extraction of dynein from yeast (Figure 2), as the final filtrate collected in step 2.3.7 showed a clear, sharp band at the position of ~350 kDa. As expected, this dynein band was absent from the flowthrough and wash that removed unwanted proteins, and the beads from which dynein was cleaved. The observation suggests that t...

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 ...

production-dynein-kinesin-motor-ensembles-on-dna-origami

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Here, we describe a protocol to create discrete and accurate inorganic nanostructures on substrates using DNA origami shapes as guiding templates. The method is demonstrated by creating plasmonic gold bowtie-shaped antennas on a transparent substrate (sapphire).

DNA Origami-Mediated Substrate Nanopatterning of Inorganic Structures for Sensing Applications

The bi-directional mitotic kinesin-5 Cin8 accumulates in clusters that split and merge during their motility. Accumulation in clusters also changes the velocity and directionality of Cin8. Here, a protocol for motility assays with purified Cin8-GFP and analysis of motile properties of single molecules and clusters of Cin8 is described.

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

Here, we present a protocol for reconstituting microtubule bundles in vitro and directly quantifying the forces exerted within them using simultaneous optical trapping and total internal reflection fluorescence microscopy. This assay allows for nanoscale-level measurement of the forces and displacements generated by protein ensembles within active microtubule networks.

Directly Measuring Forces Within Reconstituted Active Microtubule Bundles

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

Learn to produce dynein and kinesin motor ensembles on DNA origami nanostructures for single molecule observation. This protocol enables precise control over motor placement for studying cooperative transport mechanisms.

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

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