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&deg;C for future use. The BRB80 tube currently being used can be stored at 4&deg;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 &mu;L volume) into 0.5 mL microcentrifuge tubes and store at -20 &deg;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&#8722;1cm&#8722;1).</li>
	<li>Aliquot (10 &mu;L volume) into 0.5 mL microcentrifuge tubes and store at -20 &deg;C for future use.</li>
</ul>
4. Dimethyl sulfoxide, DMSO
<ul>
	<li>Aliquot (10 &mu;L volume) into 0.5 mL microcentrifuge tubes and store at -20 &deg;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 &mu;L volume) into 0.5 mL microcentrifuge tubes and store at -20 &deg;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 &mu;L volume) into 0.5 mL microcentrifuge tubes and store at -20 &deg;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 &mu;L volume) into 0.5 mL microcentrifuge tubes and store at -20 &deg;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 &mu;L volume) into 0.5 mL microcentrifuge tubes and store at -20 &deg;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 &mu;L volume) into 0.5 mL microcentrifuge tubes and store at -20 &deg;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&#8722;1cm&#8722;1).</li>
<li>Aliquot (20 &mu;L volume) into 0.5 mL microcentrifuge tubes and store at -20&deg;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 &mu;m and 0.2 &mu;m 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 &mu;L volume) into 0.5 mL microcentrifuge tubes and store at -20 &deg;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 &mu;M and store at room temperature. This solution is used to stabilize microtubules.</li>
</ul>
4. BRB80CT
<ul>
	<li>Prepare 10 &mu;M 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 &mu;g/mL glucose oxidase, 8 &mu;g/mL catalase, 10 mM DTT, and 20 &mu;M 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 &mu;L each) in 0.5 mL microcentrifuge tubes and store at -80&deg;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 &mu;L of growth solution: 4 mM MgCl2, 1 mM GTP and 5% DMSO (v/v) in BRB80 buffer.</li>
	<li>Add 6.25 &mu;L of this solution to a 20 &mu;g aliquot of lyophilized biotinylated tubulin.</li>
	<li>Vortex, then place in a heat bath at 37&deg;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 &mu;m high, and has a volume of approximately 20 &mu;L. 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="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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomolecular+motors+at+the+intersection+of+nanotechnology+and+polymer+science.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+of+microtubule+movement+driven+by+single+kinesin+molecules.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millisecond+curing+time+of+a+molecular+adhesive+causes+velocity-dependent+cargo-loading+of+molecular+shuttles.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stretching+and+Transporting+DNA+Molecules+Using+Motor+Proteins.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+transport+of+nanocrystal+CdSe+quantum+dot+nanocomposites+using+microtubules+and+kinesin+motor+proteins.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinesin+takes+one+8-nm+step+for+each+ATP+that+it+hydrolyzes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+performance+of+protein-resisting+surfaces+at+ultra-low+protein+coverages+using+kinesin+motor+proteins+as+probes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+Microtubules+Break+Up+Under+Illumination.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifetime+of+biomolecules+in+hybrid+nanodevices.">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...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Adenosine-5&#x2019;-triphosphate (ATP)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A1049</td>
<td>&nbsp;</td>
<tr>
<td>Biotin tubulin</td>
<td>&nbsp;</td>
<td>Cytoskeleton Inc.</td>
<td>T333</td>
<td>&nbsp;</td>
<tr>
<td>Casein</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>C-0376</td>
<td>&nbsp;</td>
<tr>
<td>Catalase</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>C-9322</td>
<td>&nbsp;</td>
<tr>
<td>D-(+)-Glucose</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G-7528</td>
<td>&nbsp;</td>
<tr>
<td>Dimethylsulfoxide (DMSO)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D-8779</td>
<td>&nbsp;</td>
<tr>
<td>Dithiotreitol (DTT)</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>161-0610</td>
<td>&nbsp;</td>
<tr>
<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#x2032;,N&#x2032;-tetraacetic acid (EGTA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>E-4378</td>
<td>&nbsp;</td>
<tr>
<td>FluoSpheres Biotinylated microspheres, 40 nm, yellow-green fluorescent (505/515)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>F-8766</td>
<td>&nbsp;</td>
<tr>
<td>Glucose oxidase</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G-7016</td>
<td>&nbsp;</td>
<tr>
<td>Guanosine-5&#x2019;-triphosphate (GTP)</td>
<td>&nbsp;</td>
<td>Roche Diagnostic</td>
<td>106399</td>
<td>&nbsp;</td>
<tr>
<td>Magnesium Chloride (MgCl2)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>63069</td>
<td>&nbsp;</td>
<tr>
<td>Paclitaxel (Taxol)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T1912</td>
<td>&nbsp;</td>
<tr>
<td>1,4-Piperazinediethanesulfonic acid, Piperazine-1,4-bis(2-ethanesulfonic acid), Piperazine-N,N&#x2032;-bis(2-ethanesulfonic acid) (PIPES)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P-6757</td>
<td>&nbsp;</td>
<tr>
<td>Potassium hydroxide (KOH)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P-6310</td>
<td>&nbsp;</td>
<tr>
<td>Sodium hydroxide (NaOH)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>480878</td>
<td>&nbsp;</td>
<tr>
<td>Streptavidin Alexa Fluor 568 conjugate</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>S11226</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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.

Watch this Scientific Journal Video about Cargo Loading onto Kinesin Powered Molecular Shuttles at JoVE.com

Cargo Loading onto Kinesin Powered Molecular Shuttles

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 ...

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

Research

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Biology

10.4K 조회수.  University of Florida. 표면 준수 kinesin 모터 단백질이 nanoscale 교통 시스템으로 제공할 수에 글라이딩 기능화 microtubules로 구성된 분자 셔틀. 여기 전형적인 셔틀 시스템의 조립이 설명되어 있습니다.

비디오: Cargo Loading onto Kinesin Powered Molecular Shuttles

Ex vivo Mechanical Loading of Tendon

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37&deg;C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.

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

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

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.

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

Molecular Evolution of the Tre Recombinase

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. 
We started with Cre, a 38-kDa recombinase, that recognizes a 34-bp double-stranded DNA sequence known as loxP. Because Cre can effectively eliminate genomic sequences, we set out to tailor a recombinase that could remove the sequence between the 5'-LTR and 3'-LTR of an integrated HIV-1 provirus. As a first step we identified sequences within the LTR sites that were similar to loxP and tested for recombination activity. Initially Cre and mutagenized Cre libraries failed to recombine the chosen loxLTR sites of the HIV-1 provirus. As the start of any directed molecular evolution process requires at least residual activity, the original asymmetric loxLTR sequences were split into subsets and tested again for recombination activity. Acting as intermediates, recombination activity was shown with the subsets. Next, recombinase libraries were enriched through reiterative evolution cycles. Subsequently, enriched libraries were shuffled and recombined. The combination of different mutations proved synergistic and recombinases were created that were able to recombine loxLTR1 and loxLTR2. This was evidence that an evolutionary strategy through intermediates can be successful. After a total of 126 evolution cycles individual recombinases were functionally and structurally analyzed. The most active recombinase -- Tre -- had 19 amino acid changes as compared to Cre. Tre recombinase was able to excise the HIV-1 provirus from the genome HIV-1 infected HeLa cells (see "HIV-1 Proviral DNA Excision Using an Evolved Recombinase", Hauber J., Heinrich-Pette-Institute for Experimental Virology and Immunology, Hamburg, Germany). 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.

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.

Here we report the generation of Tre recombinase through directed, molecular evolution. Tre recombinase recognizes a pre-defined target sequence within ...

From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF plates to feeder cell-free Matrigel plates.


This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 2 of 3.

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF ...

From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated.

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 3 of 3.

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

Generating a &#34;Humanized&#34; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper distribution of the genetic material requires that sister kinetochores on every chromosome become bioriented by attaching to microtubules from opposite spindle poles before progressing into anaphase. However, erroneous, non-bioriented attachment states are common and cellular pathways exist to both detect and correct such attachments during cell division. The process by which improper kinetochore-microtubule interactions are destabilized is referred to as error correction. To study error correction in living cells, incorrect attachments are purposely generated via chemical inhibition of kinesin-5 motor, which leads to monopolar spindle assembly, and the transition from mal-orientation to biorientation is observed following drug washout. The large number of chromosomes in many model tissue culture cell types poses a challenge in observing individual error correction events. Drosophila S2 cells are better subjects for such studies as they possess as few as 4 pairs of chromosomes. However, small molecule kinesin-5 inhibitors are ineffective against Drosophila kinesin-5 (Klp61F). Here we describe how to build a Drosophila cell line that effectively replaces Klp61F with human kinesin-5, which renders the cells sensitive to pharmacological inhibition of the motor and suitable for use in the cell-based error correction assay.

Generating a &quot;Humanized&quot; Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

This protocol describes how to generate a Drosophila S2 cell line that is sensitive to small molecule inhibitors of kinesin-5. The use of these cells in a cell-based error correction assay is also outlined.

Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper ...

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear transcription factor c-MYC for proteosomal degradation in the cytoplasm. The method described here involves the study of a motorless KIF5B mutant for fluorescence microscopy. The wild-type and motorless KIF5B proteins are tagged with the fluorescent protein tdTomato. The wild-type tdTomato-KIF5B appears homogenously in the cytoplasm, while the motorless tdTomato-KIF5B mutant forms aggregates in the cytoplasm. Aggregation of the motorless KIF5B mutant induces aggregation of its cargo c-MYC in the cytoplasm. Hence, this method provides a visual means to identify the cargos of Kinesin-1. A similar strategy can be utilized to identify cargos of other motor proteins.

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.

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear ...