1Department of Materials Science and Engineering, University of Florida, 2Department of Biomedical Engineering, Columbia University
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Jeune-Smith, Y., Agarwal, A., Hess, H. Cargo Loading onto Kinesin Powered Molecular Shuttles. J. Vis. Exp. (45), e2006, doi:10.3791/2006 (2010).
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.
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)
2. Magnesium Chloride, MgCl2 (100 mM in dd water)
3. Guanosine-5'-triphosphate, disodium salt, GTP (25 mM in dd water, pH adjusted to 7 by NaOH)
4. Dimethyl sulfoxide, DMSO
5. Taxol (1 mM in DMSO)
6. D-(+)-Glucose, (2 M in dd water)
7. Glucose Oxidase, (2 mg/mL in BRB80)
8. Dithiothreitol, DTT (1 M in dd water)
9. Catalase, (0.8 mg/mL in BRB80)
10. Adenosine-5'-triphosphate, ATP (100 mM in 100mM MgCl2)
11. Casein solution (20 mg/mL casein in BRB80)
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.
3.) Kinesin Preparation
4.) Microtubule Preparation
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.
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.
Figure 1. Schematic drawing of the molecular shuttles.
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% β-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: microtubules and kinesin.8 This effect is even more pronounced if the flow cell contains polymers with high oxygen diffusivity such as the polystyrene nanospheres.9
No conflicts of interest declared.
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.
|Biotin tubulin||Cytoskeleton, Inc.||T333|
|Ethylene glycol-bis(2-aminoethylether)-N,N,N€²,N€²-tetraacetic acid (EGTA)||Sigma-Aldrich||E-4378|
|FluoSpheres Biotinylated microspheres, 40 nm, yellow-green fluorescent (505/515)||Invitrogen||F-8766|
|Guanosine-5’-triphosphate (GTP)||Roche Group||106399|
|Magnesium Chloride (MgCl2)||Sigma-Aldrich||63069|
|1,4-Piperazinediethanesulfonic acid, Piperazine-1,4-bis(2-ethanesulfonic acid), Piperazine-N,N€²-bis(2-ethanesulfonic acid) (PIPES)||Sigma-Aldrich||P-6757|
|Potassium hydroxide (KOH)||Sigma-Aldrich||P-6310|
|Sodium hydroxide (NaOH)||Sigma-Aldrich||480878|
|Streptavidin Alexa Fluor 568 conjugate||Invitrogen||S11226|