Name: production of dynein and kinesin motor ensembles on dna origami nanostructures for single molecule observation
Uploaded: 2019-10-15T15:00:00
Description: Watch this Scientific Journal Video about Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation at JoVE.com

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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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>Centrifugal Filter Unit</td>
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			<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>
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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>
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			<td>Sephacryl S-500 HR</td>
			<td>GE Healthcare</td>
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			<td>Streptavidin</td>
			<td>Thermo Fisher</td>
			<td>434302</td>
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			<td>SYBR Safe DNA stain</td>
			<td>Invitrogen</td>
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			<td>Tubulin protein (&#62;99% pure): porcine brain</td>
			<td>Cytoskeleton.com</td>
			<td>T240-B</td>
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			<td>Tubulin, HiLyte 647</td>
			<td>Cytoskeleton.com</td>
			<td>TL670M-A</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 ...

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A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, ...

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...