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.

1. Growth, expression and harvesting of motor proteins controlled by a galactose induced promoter
<ol>
	<li>Using a yeast-peptone-dextrose (YPD) culture plate and a sterile inoculating stick, streak desired frozen yeast strain and incubate for 3-4 days at 30 &#176;C.</li>
	<li>Day 1 of culture growth: In the afternoon, add 10 mL of YP culture media with 2% dextrose to a 1&#34; diameter glass culture tube and inoculate it with a single colony from the plate. Grow in a rapidly rotating roller drum at 30 &#176;C overnight...

<ol>
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W., Constantinou, P. E., Kenneth Jamison, D., Diehl, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+interference+dominates+collective+transport+of+kinesin+motors+in+the+absence+of+load.">Negative interference dominates collective transport of kinesin motors in the absence of load.</a> Physical Chemistry Chemical Physics. 11 (24), 4882-4889 (2009).</li><li>Belyy, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mammalian+dynein-dynactin+complex+is+a+strong+opponent+to+kinesin+in+a+tug-of-war+competition.">The mammalian dynein-dynactin complex is a strong opponent to kinesin in a tug-of-war competition.</a> Nature Cell Biology. 18 (9), 1018-1024 (2016).</li><li>Roberts, A. J., Goodman, B. S., Reck-Peterson, S. 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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+coordination+in+motor+ensembles+revealed+using+engineered+artificial+myosin+filaments.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spherical+DNA+Origami+as+a+Programmable+Cargo+Structure+for+Investigating+the+Emergent+Motility+of+Dynein+and+Kinesin+Ensembles.">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. 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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.

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

Watch this Scientific Journal Video about Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation at JoVE.com

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

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 ...

Our protocol is designed to probe the biophysical mechanisms governing the transport of cargo by teams of identical or oppositely directed cytoskeletal motor proteins. By using DNA origami for molecular construction, this method can choose for the type, number, and location of motors on precisely defined cargo shapes. While this protocol focuses on the microtubule-based motors dynein and kinesin, it is adaptable to the actin-based motor myosin, as well as to other protein systems that function cooperatively.One challenging aspect of this protocol is to maintain the integrity and activity of the motor proteins throughout the purification process, as they're sensitive to heat and mechanical force. To induce motor protein growth and expression via galactose promoter, use a sterile inoculation wand to streak the frozen yeast strain of interest on a yeast-peptone-dextrose culture plate for a three-to four-day incubation at 30 degrees Celsius. On day four of culture, when the optical density at 600 nanometers is between 1.5 and two, collect the yeast cells by centrifugation, and resuspend the pellet in water to wash them and consolidate the cells in a single bottle.Spin the cells again for collection, and resuspend the pellet in about two milliliters of double-distilled water. Then use a 10-milliliter pipette to slowly dispense the cell slurry into liquid nitrogen one drop at a time to produce frozen yeast cell pellets. For motor protein purification, use a blade-type coffee grinder pre-chilled with liquid nitrogen to grind the frozen yeast pellets into a fine powder, and transfer the yeast powder into a pre-chilled, 100-milliliter, glass beaker on ice.Add a small volume of freshly prepared 4X lysis buffer with supplements to the powder so that the final concentration of the buffer does not exceed 1X, and place the beaker in a 37-degree Celsius water bath with continuous stirring with a spatula to quickly thaw the powder. Add additional 4X buffer with supplements to bring the final buffer concentration to 1X in the lysate. The final volume is often between 25 and 30 milliliters.Then collect the yeast cells by centrifugation, and transfer the soluble protein containing supernatant into a new 50-milliliter conical tube on ice. For IgG affinity purification, add 200 microliter of washed affinity bead suspension to the protein extract, and incubate the mixture at four degrees Celsius for one hour with gentle rotation. At the end of the incubation, filter the mixture through the same chromatography column used to prepare the beads at four degrees Celsius, followed by two washes with five milliliters of wash buffer per wash on ice.After the second wash, rinse the beads on ice with five milliliters of TEV buffer, allowing the buffer to fully drain from the column. For DNA oligonucleotide labeling, cap the chromatography column, and incubate the motor beads with 100 microliters of TEV buffer supplemented with 10 to 20 micromolar of the purified benzylguanine oligo at room temperature for 10 to 15 minutes, gently resuspending the beads once a minute. At the end of the incubation, wash the beads four times with fresh TEV buffer per wash as just demonstrated, before recapping the bottom of the tube to allow resuspension of the motor beads in no more than 200 microliters of fresh TEV buffer.For TEV cleavage, transfer the motor bead suspension to a two-milliliter, round-bottom, microcentrifuge tube, and incubate the motor beads with approximately 0.3 units of TEV protease per microliter of motor bead mixture at 16 degrees Celsius for one hour with gentle rotation. At the end of the incubation, centrifuge the motor beads, and collect the mixture at the bottom of the tube. Next, use a cut P1000 pipette tip to transfer the mixture to a spin column, and centrifuge the mixture as just demonstrated.Collect the filtrate containing the TEV-cleaved, purified motors, and aliquot the filtrate in 50-microliter volumes for microtubule affinity purification. Then flash freeze the aliquots in liquid nitrogen for minus 80-degree Celsius storage. For chassis purification, in the late afternoon of the day before the purification, gently layer 80 microliters each of concentration of glycerol in origami folding buffer in a centrifuge tube.The boundaries between the layers should be slightly visible. After an overnight incubation at four degrees Celsius, layer 10%glycerol in origami folding buffer in folded chassis solution over the top layer of the gradient, and centrifuge the gradient with the chassis. At the end of the centrifugation, collect 50-microliter fractions of the gradient in a top-to-bottom direction, and load five microliters of each fraction into individual wells of a 2%agarose gel.After 90 to 120 minutes at 70 volts, image the gel using conditions suitable for the DNA gel stain used. Quantified concentrations of the selected fractions of interest can be determined using the appropriate standard spectroscopic methods. SDS-PAGE analysis can be used to confirm the successful extraction of dynein from yeast, as the final filtrate shows a clear, sharp band at approximately 350 kilodaltons.TEV protease is also present in the final filtrate and forms a clear band at about 50 kilodaltons. After microtubule affinity purification, dynein appears as a clear, single band at 350 kilodaltons, while kinesin can be observed as slightly smearing, multiple bands at about 120 kilodaltons. In addition to the TEV protease, a noticeable amount of tubulin can also be observed in the final supernatant.The folding of DNA origami structures can be assayed by agarose gel electrophoresis, with a shift in mobility between the pure unfolded scaffold strand and the folding reaction indicating origami folding. Additionally, the folding reaction indicates the presence of some multimerization of chassis structures. The motility of motor chassis ensembles is easily detectable and measurable on kymographs generated from TIRF movies, as the kymographs of flexible chassis conjugated to seven dynein proteins demonstrate highly processive runs at relatively consistent velocities.It is vital to carefully control the temperature of the motor proteins at each step of the procedure. Following the purification of the motor proteins and DNA origami chassis, the two components can be conjugated for motility assays of the ensembles under a TIRF microscope. This method enables the biophysical and biochemical mechanisms that govern the emergent motility of the motor ensembles to be deciphered.

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

Research

JoVE Journal

Biochemistry

6.4K Görüntüleme.  Smith College. Protokolümüz, aynı veya zıt yönlendirilmiş sitosiskelet elmotor proteinlerinden oluşan ekipler tarafından kargonun taşınmasını yöneten biyofiziksel mekanizmaları araştırmak için tasarlanmıştır. Moleküler yapı için DNA origami kullanarak, bu yöntem, tam olarak tanımlanmış kargo şekilleri üzerinde motorların türü, sayısı ve konumu için seçebilirsiniz. Bu protokol mikrotübül bazlı motorlar dynein ve kinesin üzerinde duruluyor iken, aktin tabanlı motor miy...