Name: Cargo Loading onto Kinesin Powered Molecular Shuttles (Video) | JoVE Journal
Uploaded: 2010-11-03T17:00:00.000+00:00
Duration: 9 min
Description: Learn to load nanospheres onto kinesin-powered microtubules for nanoscale transport using an inverted motility assay. This method enables researchers to study molecular shuttle systems for targeted drug delivery applications.

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5 minutes

We are heavily indebted to Jonathon Howard, whose group developed the basic protocol for a gliding motility assay which was subsequently adapted by us. Financial support from NSF grant DMR0645023 is gratefully acknowledged.

1.) Buffers and Reagents
These solutions should be prepared in advance and stored in conveniently sized aliquots. An aliquot should contain sufficient solution for a typical experiment and a fresh aliquot should be used for each motility assay. The storage conditions and typical aliquot sizes are also mentioned in the following protocols.
1. BRB80 buffer, (80 mM PIPES, 1 mM MgCl2, 1 mM EGTA in deionized distilled (dd) water, pH adjusted to 6.9 by KOH)
<ul>
	<li>Make up a 100 mL stock solution of 0.5 M EGTA in dd water. Adjust pH to 7.0 using 2 M NaOH solution.</li>
	<li>Make up a 100 mL stock solution of 1 M MgCl2 in dd water. Autoclave the solution.</li>
	<li>Add 24.2 g of PIPES and 3.1 g KOH pellets in approximately 800 mL of dd water and stir to dissolve. Adjust pH to 6.9 using 1 M KOH solution. Add 2 mL of 0.5 M EGTA stock solution and 1 mL of 1 M MgCl2 stock solution. Bring up the volume to 1000 mL with dd water.</li>
	<li>Aliquot into 50 mL falcon tubes and freeze at -20°C for future use. The BRB80 tube currently being used can be stored at 4°C or at room temperature.</li>
</ul>
2. Magnesium Chloride, MgCl2 (100 mM in dd water)
<ul>
	<li>Dilute in dd water to achieve a final concentration of 100 mM.</li>
	<li>Aliquot (10 μL volume) into 0.5 mL microcentrifuge tubes and store at -20 °C for future use</li>
</ul>
3. Guanosine-5'-triphosphate, disodium salt, GTP (25 mM in dd water, pH adjusted to 7 by NaOH)
<ul>
	<li>Weigh out and dissolve in dd water and adjust the pH to 7 by 2 M NaOH solution.</li>
	<li>Verify concentration by measuring UV absorbance at 260 nm. (Use an extinction coefficient of 11.7 x 103 M−1cm−1).</li>
	<li>Aliquot (10 μL volume) into 0.5 mL microcentrifuge tubes and store at -20 °C for future use.</li>
</ul>
4. Dimethyl sulfoxide, DMSO
<ul>
	<li>Aliquot (10 μL volume) into 0.5 mL microcentrifuge tubes and store at -20 °C for future use.</li>
</ul>
5. Taxol (1 mM in DMSO)
<ul>
	<li>Weigh out and dissolve in DMSO under fume hood to achieve a final concentration of 1 mM.</li>
	<li>Aliquot (20 μL volume) into 0.5 mL microcentrifuge tubes and store at -20 °C for future use.</li>
</ul>
6. D-(+)-Glucose, (2 M in dd water)
<ul>
	<li>Weigh out and dissolve in dd water to achieve a final concentration of 2 M.</li>
	<li>Aliquot (20 μL volume) into 0.5 mL microcentrifuge tubes and store at -20 °C for future use.</li>
</ul>
7. Glucose Oxidase, (2 mg/mL in BRB80)
<ul>
	<li>Dissolve in BRB80 to achieve a final concentration of 2 mg/mL.</li>
	<li>Aliquot (20 μL volume) into 0.5 mL microcentrifuge tubes and store at -20 °C for future use.</li>
</ul>
8. Dithiothreitol, DTT (1 M in dd water)
<ul>
	<li>Dissolve in dd water under fume hood to achieve a final concentration of 1 M.</li>
	<li>Aliquot (20 μL volume) into 0.5 mL microcentrifuge tubes and store at -20 °C for future use.</li>
</ul>
9. Catalase, (0.8 mg/mL in BRB80)
<ul>
	<li>Dissolve in BRB80 in at least 2 stages to achieve a final concentration of 0.8 mg/mL. Determine the concentration at each stage by measuring UV absorbance at 276 nm and 406 nm (Use an extinction coefficient of 3.1 x 105 M-1 cm-1 at 276 nm and 2.2 x 105 M-1 cm-1 at 406 nm and the equation A=cL).</li> 
<li>Aliquot (20 μL volume) into 0.5 mL microcentrifuge tubes and store at -20 °C for future use.</li>
</ul>
10. Adenosine-5'-triphosphate, ATP (100 mM in 100mM MgCl2)
<ul>
<li>Prepare a stock solution of 100 mM MgCl2 in dd water. Weigh out dry powder and dissolve in this stock solution to achieve a final concentration of 100mM.</li>
<li>Verify concentration by measuring UV absorbance at 260 nm. (Use an extinction coefficient of 15.4 x 103 M−1cm−1).</li>
<li>Aliquot (20 μL volume) into 0.5 mL microcentrifuge tubes and store at -20°C for future use.</li>
</ul>
11. Casein solution (20 mg/mL casein in BRB80)
<ul>
<li>Add approximately 3 g casein to 30 mL dd water in a 50 mL falcon tube. Vortex for approximately 1 hour until solution develops thick consistency.</li>
<li>Centrifuge at approximately 15000 g for 30 minutes. Filter the supernatant through 0.5 μm and 0.2 μm syringe filters.</li>
<li>Determine the concentration of the supernatant by measuring UV absorbance at 280 nm (Use an extinction coefficient of 0.67 mL mg-1 cm-1). Dilute it to 20 mg/mL in BRB80.</li>
<li>Aliquot (20 μL volume) into 0.5 mL microcentrifuge tubes and store at -20 °C for future use. </li>
</ul>
2.) Standard Solutions
These are prepared on the day of the experiment and should be discarded after the experiment is over. Prepare 1 mL of each.
1. BRB80CS0.5
<ul>
	<li>Dilute casein solution in BRB80 to a final concentration of 0.5 mg/mL and store over ice. This solution is introduced into the flow cell prior to kinesin and helps retain kinesin activity after surface adsorption.</li>
</ul>
2. BRB80CA
<ul>
	<li>Prepare 0.2 mg/mL casein and 1 mM ATP in BRB80 and store over ice. Kinesin is further diluted using this solution before introduction into the flow cell.</li>
</ul>
3. BRB80T
<ul>
	<li>Dilute taxol solution in BRB80 to a final concentration of 10 μM and store at room temperature. This solution is used to stabilize microtubules.</li>
</ul>
4. BRB80CT
<ul>
	<li>Prepare 10 μM taxol and 0.2 mg/mL casein in BRB80 and store at room temperature. This is used to further prepare the antifade and microtubule solutions.</li>
</ul>
5. BRB80AF
<ul>
	<li>Prepare 20 mM D-glucose, 20 μg/mL glucose oxidase, 8 μg/mL catalase, 10 mM DTT, and 20 μM ATP in BRB80CT and store at room temperature. This solution is used to dilute streptavidin and nanospheres and "wash out" the excess streptavidin in the flow cell. The kinesin speed can be controlled by adjusting the ATP concentration in this solution.</li>
</ul>
3.) Kinesin Preparation
<ul>
	<li>Express a kinesin construct consisting of the wild-type, full-length Drosophila melanogaster kinesin heavy chain and a C-terminal His-tag in Escherichia coli and purify using a Ni-NTA column as described in 6.</li> 
<li>Make aliquots (10 μL each) in 0.5 mL microcentrifuge tubes and store at -80°C for future use. The concentration of active kinesin in these aliquots is approximately 200 nM.7</li>
	<li>For a typical experiment, dilute the kinesin solution 20 fold in BRB80CA. Label the solution KIN20 and store over ice.</li>
</ul>
4.) Microtubule Preparation
<ul>
	<li>In a 0.5 mL microcentrifuge tube, prepare 25 μL of growth solution: 4 mM MgCl2, 1 mM GTP and 5% DMSO (v/v) in BRB80 buffer.</li>
	<li>Add 6.25 μL of this solution to a 20 μg aliquot of lyophilized biotinylated tubulin.</li>
	<li>Vortex, then place in a heat bath at 37°C for 30 minutes to polymerize. Dilute 100-fold in BRB80T and vortex gently. Label the solution MT100 and store at room temperature.</li>
	<li>Make a 10 fold dilution of MT100 in BRB80AF. Label the solution MT1000.</li>
</ul>
5.) Streptavidin and Nanosphere Solution
Prepare AlexaFluor568-labeled streptavidin at a concentration of 100 nM in BRB80AF. Label it STV100 and store over ice. Similarly, dilute nanospheres 5000 fold in BRB80AF solution. Label it NS5000 and store over ice.
6.) Flow Cell Construction
Construct a flowcell using two glass coverslips separated by double-sided tape. This flow cell is approximately 2 cm long, 1 cm wide and 100 μm high, and has a volume of approximately 20 μL. Solutions are introduced into the flow cell from one side using a pipette and wicked out from the other using filter paper.
7.) Inverted Assay Assembly
Glass surface is first coated with casein which allows kinesin to retain its functionality upon adsorption. After kinesin is adsorbed, microtubules are introduced, which are held by kinesin. Microtubules are then coated with fluorescent streptavidin. After washing out the excess streptavidin, biotinylated polystyrene fluorescein nanospheres (40 nm diameter) are introduced. Surface adsorbed stationary nanospheres collide with the moving microtubules and get loaded onto them (Figure 1).
The order of flow of solutions and time allowed before the introduction of the next solution are listed below.
<ul>
	<li>BRB80CS0.5, 5 minutes</li>
	<li>KIN20, 5 minutes</li>
	<li>MT1000, 5 minutes</li>
	<li>STV100, 5 minutes</li>
	<li>BRB80AF, 3x</li>
	<li>NS5000</li>
</ul>
8.) Microscopy
Mount the flow cell on the microscope stage immediately after nanosphere introduction. In this experiment, an Eclipse TE2000-U fluorescence microscope (Nikon, Melville, NY) equipped with a 100X oil objective (N.A. 1.45), an X-cite 120 lamp (EXFO, Ontario, Canada) and an iXon EMCCD camera (ANDOR, South Windsor, CT) was used. A FITC filter cube (#48001) and a TRITC filter cube (#48002, Chroma Technologies, Rockingham, VT) were used to image nanospheres and microtubules respectively on the bottom surface of flow cells. The exposure time was 0.2s, while the time between exposures was 2 s.
<img src="/files/ftp_upload/2006/2006fig1.jpg" alt="Biotin-streptavidin nanosphere binding on microtubule in kinesin-powered transport diagram." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. Schematic drawing of the molecular shuttles.

<ol><li>Agarwal, A., Hess, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biomolecular+motors+at+the+intersection+of+nanotechnology+and+polymer+science%5BTitle%5D)+AND+%22Progress+in+Polymer+Science%22%5BJournal%5D)">Biomolecular motors at the intersection of nanotechnology and polymer science.</a> Progress in Polymer Science. 35 (1-2), 252-252 (2010).</li>
<li>Howard, J., Hunt, A. J., Baek, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assay+of+microtubule+movement+driven+by+single+kinesin+molecules%5BTitle%5D)+AND+%22Methods+Cell+Biol%22%5BJournal%5D)">Assay of microtubule movement driven by single kinesin molecules.</a> Methods Cell Biol. 39, 137-137 (1993).</li>
<li>Agarwal, A., Katira, P., Hess, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Millisecond+curing+time+of+a+molecular+adhesive+causes+velocity-dependent+cargo-loading+of+molecular+shuttles%5BTitle%5D)+AND+%22Nano+Lett%22%5BJournal%5D)">Millisecond curing time of a molecular adhesive causes velocity-dependent cargo-loading of molecular shuttles.</a> Nano Lett. 9 (3), 1170-1170 (2009).</li>
<li>Diez, S., Reuther, C., Dinu, C., Seidel, R., Mertig, M., Pompe, W., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stretching+and+Transporting+DNA+Molecules+Using+Motor+Proteins%5BTitle%5D)+AND+%22Nano+Lett%22%5BJournal%5D)">Stretching and Transporting DNA Molecules Using Motor Proteins.</a> Nano Lett. 3 (9), 1251-1251 (2003).</li>
<li>Bachand, G. D., Rivera, S. B., Boal, A. K., Gaudioso, J., Liu, J., Bunker, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assembly+and+transport+of+nanocrystal+CdSe+quantum+dot+nanocomposites+using+microtubules+and+kinesin+motor+proteins%5BTitle%5D)+AND+%22Nano+Lett%22%5BJournal%5D)">Assembly and transport of nanocrystal CdSe quantum dot nanocomposites using microtubules and kinesin motor proteins.</a> Nano Lett. 4 (5), 817-817 (2004).</li>
<li>Coy, D. L., Wagenbach, M., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Kinesin+takes+one+8-nm+step+for+each+ATP+that+it+hydrolyzes%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">Kinesin takes one 8-nm step for each ATP that it hydrolyzes.</a> J. Biol. Chem. 274 (6), 3667-3667 (1999).</li>
<li>Katira, P., Agarwal, A., Fischer, T., Chen, H. -Y., Jiang, X., Lahann, J., Hess, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantifying+the+performance+of+protein-resisting+surfaces+at+ultra-low+protein+coverages+using+kinesin+motor+proteins+as+probes%5BTitle%5D)+AND+%22Advanced+Materials%22%5BJournal%5D)">Quantifying the performance of protein-resisting surfaces at ultra-low protein coverages using kinesin motor proteins as probes.</a> Advanced Materials. 19, 3171-3171 (2007).</li>
<li>Vigers, G. P. A., Coue, M., McIntosh, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluorescent+Microtubules+Break+Up+Under+Illumination%5BTitle%5D)+AND+%22J.+Cell+Biol%22%5BJournal%5D)">Fluorescent Microtubules Break Up Under Illumination.</a> J. Cell Biol. 107, 1011-1011 (1988).</li>
<li>Brunner, C., Hess, H., Ernst, K. -H., Vogel, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lifetime+of+biomolecules+in+hybrid+nanodevices%5BTitle%5D)+AND+%22Nanotechnology%22%5BJournal%5D)">Lifetime of biomolecules in hybrid nanodevices.</a> Nanotechnology. 15 (10), S540-S540 (2004).</li>
</ol>

No conflicts of interest declared.

With minor modifications, this protocol has been successfully used by a variety of groups to assemble kinesin-microtubule based motility assays. 10 mM DTT in the final motility solution can be replaced with 0.5% &beta;-mercaptoethanol. Standard solutions (BRB80AF, KIN20 and MT1000) more than 2 hours old should not used. Any solution containing taxol and especially microtubules should never be placed on ice. Excessive exposure of the flow cell to UV excitation light results in photodamage to the functional components: mic...

<table><tbody><tr><td>Adenosine-5&amp;rsquo;-triphosphate (ATP)</td><td>Invitrogen</td><td>A1049</td><td></td></tr><tr><td>Biotin tubulin</td><td>Cytoskeleton, Inc.</td><td>T333</td><td></td></tr><tr><td>Casein</td><td>Sigma-Aldrich</td><td>C-0376</td><td></td></tr><tr><td>Catalase</td><td>Sigma-Aldrich</td><td>C-9322</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G-7528</td><td></td></tr><tr><td>Dimethylsulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D-8779</td><td></td></tr><tr><td>Dithiotreitol (DTT)</td><td>Bio-Rad</td><td>161-0610</td><td></td></tr><tr><td>Ethylene glycol-bis(2-amin&amp;#8211;thylether)-&lt;em&gt;N,N,N&amp;#8242;,N&amp;#8242;&lt;/em&gt;-tetraacetic acid (EGTA)</td><td>Sigma-Aldrich</td><td>E-4378</td><td></td></tr><tr><td>FluoSpheres Biotinylated microspheres, 40 nm, yellow-green fluorescent (505/515)</td><td>Invitrogen</td><td>F-8766</td><td></td></tr><tr><td>Glucose oxidase</td><td>Sigma-Aldrich</td><td>G-7016</td><td></td></tr><tr><td>Guanosine-5&amp;rsquo;-triphosphate (GTP)</td><td>Roche Group</td><td>106399</td><td></td></tr><tr><td>Magnesium Chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>63069</td><td></td></tr><tr><td>Paclitaxel (Taxol)</td><td>Sigma-Aldrich</td><td>T1912</td><td></td></tr><tr><td>1,4-Piperazinediethanesulfonic acid, Piperazine-1,4-bis(2-ethanesulfonic acid), Piperazine-N,N&amp;#8242;-bis(2-ethanesulfonic acid) (PIPES)</td><td>Sigma-Aldrich</td><td>P-6757</td><td></td></tr><tr><td>Potassium hydroxide (KOH)</td><td>Sigma-Aldrich</td><td>P-6310</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Sigma-Aldrich</td><td>480878</td><td></td></tr><tr><td>Streptavidin Alexa Fluor 568 conjugate</td><td>Invitrogen</td><td>S11226</td><td></td></tr></tbody></table>

cargo loading onto kinesin powered molecular shuttles

Cargo Loading onto Kinesin Powered Molecular Shuttles

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport of cargo. While kinesins tail domain binds to a variety of cargoes, kinesins head domains utilize the chemical energy stored in ATP molecules to step along the microtubule lattice. The long, stiff microtubules serve as tracks for long-distance intracellular transport.
These motors and filaments can also be employed in microfabricated synthetic environments as components of molecular shuttles 1. In a frequently used design, kinesin motors are anchored to the track surface through their tails, and functionalized microtubules serve as cargo carrying elements, which are propelled by these motors. These shuttles can be loaded with cargo by utilizing the strong and selective binding between biotin and streptavidin. The key components (biotinylated tubulin, streptavidin, and biotinylated cargo) are commercially available.
Building on the classic inverted motility assay 2, the construction of molecular shuttles is detailed here. Kinesin motor proteins are adsorbed to a surface precoated with casein; microtubules are polymerized from biotinylated tubulin, adhered to the kinesin and subsequently coated with rhodamine-labeled streptavidin. The ATP concentration is maintained at subsaturating concentration to achieve a microtubule gliding velocity optimal for loading cargo 3. Finally, biotinylated fluorescein-labeled nanospheres are added as cargo. Nanospheres attach to microtubules as a result of collisions between gliding microtubules and nanospheres adhering to the surface.
The protocol can be readily modified to load a variety of cargoes such as biotinylated DNA4, quantum dots 5 or a wide variety of antigens via biotinylated antibodies 4-6.

Molecular shuttles consisting of functionalized microtubules gliding on surface-adhered kinesin motor proteins can serve as a nanoscale transport system. Here, the assembly of a typical shuttle system is described.

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport ...

The overall goal of this procedure is to load Nanospheres onto microtubules, which are gliding on a surface coated with kinase in motors. This is accomplished by first coating the surface of a glass flow cell with casein solution. The second step of the procedure is to absorb kinesin motor proteins onto the flow cell surface.The third step of the procedure is to add the microtubules. The final step of the procedure is to add the strept haver in molecules, which will bind into the microtubules, and then add the nanospheres. The nanospheres bind into the flow cell surface where they may get picked up by the microtubules.Ultimately, one can observe how nanospheres are picked up by microtubules and are transported across the surface using fluorescence microscopy. Hi, I'm Elaine Jen Smith from the laboratory of Dr.Henry Hess and the Department of Material Science and Engineering at the University of Florida. Today we will show you a procedure for the inverted motility assay.We use this procedure in our laboratory to study loading of cargo onto microtubules and movement of molecular shuttles. So let's get started. Prior to the start of this procedure, freshly prepare all stock solutions as described in the written portion of this video.Once stock solutions have been made, begin micro tubial preparation by pipetting 25 microliters of growth solution into a 0.65 milliliter micro centrifuge tube. Then add 6.25 microliters of the growth solution to 20 micrograms of lyophilized biotinylated tubulin. Vortex the solution and place it in a heat bath at 37 degrees Celsius for 30 minutes to polymerize following incubation, dilute the solution 100 fold in Taxol Buffer and gently vortex.This solution serves to stabilize the microtubules. Label the solution as empty 100 and store it at room temperature. Next, make a tenfold dilution of MT 100 in anti fade buffer and label this solution as MT 1000.Then prepare the Kinesin motors by diluting the kinesin solution 20 fold in case in a TP buffer. Label the solution as kinase in 20 and store it on ice. Finally, make the streptavidin and nanosphere solutions.First, add Alexa Fluor 5 6 8 labeled streptavidin to a concentration of 100 nano molars in Antifa Buffer. Label the solution as STV 100, then dilute nanospheres 5, 000 fold in Antifa Buffer and label this mixture as NS 5, 000. Store both of these solutions over ice.Following solution preparation for the cargo loading assay, construct a flow cell using a cover slide and a glass cover slip separated by double-sided tape. First stick two strips of double-sided tape at the edges of a cover slide, leaving a gap of about one centimeter between the strips. Then lay a cover slip over the tape.Apply pressure to the cover slip using the flat edge of a tweezer to make a seal. The constructed flow cell is approximately two centimeters long, one centimeter wide and 100 microns high, and has a volume of approximately 20 microliters. To begin inverted assay assembly.Place the tip of the pipette between the cover slip and the slide. Then inject 0.5 milligrams per milliliter casein solution into the flow cell. This coating on the flow cell will allow kinesin to retain its functionality upon absorption.Wait five minutes for the casein solution to coat. Next, absorb Kinesin onto the flow cell by introducing the kinase in 20 solution solutions are introduced into the flow cells by placing the tip of the pipette between the cover slip and the slide. Then the solution is injected into the flow cell from one side, while it is simultaneously wicked out the other side.With filter paper, allow five minutes for the kinase in to absorb. Next, introduce the microtubules into the flow cell by pipetting the MT 1000 solution into the flow cell. Allow five minutes for microtubules to settle and attach to the kinase in motors following micro tubial attachment to kinase in motors.Coat the microtubules with fluorescent strep TTR in by introducing the STV 100 solution into the flow cell. Allow five minutes for strep TTR in molecules to bind to the microtubules. Wash out the excess strep TTR in by introducing three 30 microliter washes of Antifa solution into the flow cell.Finally, introduce the biotinylated polystyrene fluorescein nanospheres by pipetting the NS 5, 000 solution into the flow cell. The surface absorbs stationary. Nanospheres will collide with the moving microtubules and load onto them.In this experiment, an eclipse TE 2000 U fluorescence microscope equipped with a 100 x oil objective and X site, one 20 lamp and an exon E-M-C-C-D camera is used. First, apply one drop of immersion oil over the objective. Then mount the flow cell on the microscope stage using the trixy filter cube.Bring the micro tubules into focus, then switch to the fitzy filter cube to view the nanospheres. Next, open the and or I exon program and set the acquisition mode to kinetic. Use an exposure time of 0.5 seconds, a kinetic series length of 20 and a kinetic cycle time of six seconds.Switch the microscope from eyepiece to camera and begin acquiring images. For the first four images, use the trixi cube to image the microtubules. For the next 12 images, switch to the Fitz filter cube to acquire images of the nanospheres.Then switch back to the trixi filter cube. For the last four images to image the microtubules, the microtubules are observed using the Trissy filter cube, the filter cube is switched to fite To visualize the nanospheres here, nanospheres being transported by microtubules are identified by the arrows. Not all nanospheres have been picked up by microtubules.Now the location of the nanospheres is shown 36 seconds later revealing that the nanospheres have shifted. When the microtubules are imaged again, it becomes apparent that the microtubules have also traveled across the field of view. These images demonstrate that the nanospheres have been displaced due to micro tubial shuttling.We have just shown you how to perform the inverted motility assay. When doing this procedure, it's important to remember to switch the filter cubes while acquiring images so that both the microtubules and the nanospheres can be seen. So that's it.Thanks for watching and good luck with your experiments.

cargo-loading-onto-kinesin-powered-molecular-shuttles

Research

JoVE Journal

Biology

Single molecule microscopy approch provided novel insights into nuclear transport.

Single-Molecule Imaging of Nuclear Transport

Intracellular transport of cargoes, such as vesicles or organelles, is carried out by molecular motor proteins that track on polarized microtubules. This protocol describes the correlation of the directionality of transport of individual cargo particles moving inside neurons, to the relative amount and type of associated motor proteins.

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis

Here, a protocol is presented to identify Kinesin-1 cargos. A motorless mutant of the Kinesin-1 heavy chain (KIF5B) aggregates in the cytoplasm and induces aggregation of its cargos. Both aggregates are detected under fluorescence microscopy. A similar strategy can be employed to identify cargos of other motor proteins.

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

We present a protocol to build molecular shuttles, where surface-adhered kinesin motor proteins propel dye-labelled microtubules. Weak interactions of the kinesins with the surface enables their reversible attachment to it. This creates a nanoscale system which exhibits dynamic assembly and disassembly of its components while retaining its functionality.

Assembling Molecular Shuttles Powered by Reversibly Attached Kinesins

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.

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

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

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

A new in vitro system for simultaneously loading four tendons in culture is described.

Ex vivo Mechanical Loading of Tendon

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

Loading Drosophila Nerve Terminals with Calcium Indicators

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within the LTR sequences of the HIV-1 provirus, resulting in the excision and eradication of the provirus from infected human cells. While still in its infancy, directed molecular evolution will allow the creation of custom enzymes that will serve as tools of molecular surgery and molecular medicine.

Molecular Evolution of the Tre Recombinase

Learn to load nanospheres onto kinesin-powered microtubules for nanoscale transport using an inverted motility assay. This method enables researchers to study molecular shuttle systems for targeted drug delivery applications.

Cargo Loading onto Kinesin Powered Molecular Shuttles

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