The authors gratefully acknowledge financial support under NSF grant NSF-DMR 1807514. The authors thank G. Bachand and V. Vandelinder for providing the GFP-kinesin protein. This work was performed, in part, at the Center for Integrated Nanotechnologies, an Office of Science User Facility operated for the US Department of Energy (DOE) Office of Science by Los Alamos National Laboratory (contract no. DE- AC52-06NA25396) and Sandia National Laboratories (con- tract no. 97 DE-AC04-94AL85000). The authors thank Dr. Jennifer Neff and AllVivo Vascular for their gift of PEG-PPG-PEG functionalized with NTA.

1. Solution preparation
CAUTION: Three of the reagents used in this protocol (toluene, dimethyldichlorosilane, and dithiothreitol) are highly toxic. Please consult the relevant material safety data sheets (MSDS) before use. Furthermore, parts of this protocol need to be performed under a higher level of protection, wearing safety glasses and two sets of protective gloves as indicated. Unless otherwise advised, the experiments can be performed on lab benches while using all the appropriate personal protective equipment (safety glasses, gloves, lab coat, full length pants, and closed-toe shoes).
NOTE: The concentrations of the ATP, kinesin, and microtubules that are used in this protocol can be modified given the needs of each experiment. However, if modified, please ensure that the final concentrations of the other reagents remain the same as given below. All experiments were performed at room temperature, (~25 &#176;C).
<ol>
	<li>Preparation of stock solutions 
	NOTE: Prepare and aliquot the following solutions beforehand. All aliquots can be prepared at room temperature (~25 &#176;C).
	<ol>
		<li>BRB80 buffer 
		NOTE: The buffer for most of the solutions used in this protocol is BRB80. BRB80 can be prepared in large quantities and stored in a -20 &#176;C freezer.
		<ol>
			<li>Dissolve 24.2 g of piperazine-N,N&#8242;-bis(2-ethanesulfonic acid) (PIPES) and 3.1 g of potassium hydroxide (KOH) in 800 mL in deionized water to make 800 mL of 100 mM PIPES. Add 100 mL of 10 mM magnesium chloride (MgCl2) and 100 mL of 10 mM ethylene glycol tetraacetic acid (EGTA) to get a final volume of 1 L. Raising the pH to 8 with potassium hydroxide (KOH) can help in the dissolution of PIPES and EGTA. 
			NOTE: The final concentrations of chemicals in the BRB80 buffer are 80 mM PIPES, 1 mM MgCl2, and 1 mM EGTA.</li>
			<li>Adjust the pH of the buffer to 6.9 using KOH and hydrochloric acid (HCl).</li>
		</ol>
		</li>
		<li>ATP
		<ol>
			<li>Prepare a 1 mL solution of 100 mM ATP in ultrapure water. Pipet the solution into 10 &#956;L aliquots. Store the aliquots in a -80 &#176;C freezer.</li>
		</ol>
		</li>
		<li>GTP
		<ol>
			<li>Prepare a 500 &#956;L solution of 25 mM GTP in ultrapure water. Pipet the solution into 5 &#956;L aliquots. Store the aliquots in a -80 &#176;C freezer.</li>
		</ol>
		</li>
		<li>MgCl2
		<ol>
			<li>Prepare a 1 mL solution of 100 mM MgCl2 in ultrapure water. Pipet the solution into 10 &#956;L aliquots. Store the aliquots in a -20 &#176;C freezer.</li>
		</ol>
		</li>
		<li>Casein
		<ol>
			<li>Weigh 1 g of dry casein and transfer it to a 50 mL conical centrifuge tube. Add 35 mL of BRB80 buffer to the tube to dissolve the casein powder.</li>
			<li>Place the solution in a tumbler in a cold room to dissolve overnight. At this point, the solution will look thick and viscous.</li>
			<li>Leave the tube upright in a 4 &#176;C fridge to allow for any large undissolved clumps to settle. Transfer the supernatant to a new tube.</li>
			<li>Spin the tube in a centrifuge at 1,000 x g to pellet out more precipitates. Once again, transfer the supernatant to a new tube.</li>
			<li>Repeatedly filter the solution using 0.2 &#956;m (7 bar maximum pressure) filters. The solution being thick, many filters will get clogged, so repeat this step until the filter is clear and unclogged.</li>
			<li>Determine the casein concentration in the resulting solution using UV/Vis spectrophotometry, using casein&#8217;s extinction coefficient of 19 mM-1&#8729;cm-1 at 280 nm20. Assuming a molecular weight of 23 kDa for casein, dilute the solution to a concentration of 20 mg&#8729;mL-1 in BRB80.</li>
			<li>Pipet the solution into 20 &#956;L aliquots and store the aliquots in a -20 &#176;C freezer.</li>
		</ol>
		</li>
		<li>D-Glucose
		<ol>
			<li>Prepare a 1 mL solution of 2 M D-glucose in ultrapure water. Pipet the solution into 10 &#956;L aliquots. Store the aliquots in a -20 &#176;C freezer.</li>
		</ol>
		</li>
		<li>Glucose oxidase
		<ol>
			<li>Prepare a 1 mL solution of 20 mg&#8729;mL-1 glucose oxidase in BRB80. Pipet the solution into 10 &#956;L aliquots. Store the aliquots in a -20 &#176;C freezer.</li>
		</ol>
		</li>
		<li>Catalase
		<ol>
			<li>Prepare a 1 mL solution of 0.8 mg&#8729;mL-1 catalase in BRB80. Pipet the solution into 10 &#956;L aliquots. Store the aliquots in a -20 &#176;C freezer.</li>
		</ol>
		</li>
		<li>Dithiothreitol (DTT) 
		NOTE: Since DTT is slightly volatile and toxic, please perform the following steps under a fume hood.
		<ol>
			<li>Prepare a 1 mL solution of 1 M DTT diluted in ultrapure water. Pipet the solution into 10 &#956;L aliquots. Store the aliquots in a -20 &#176;C freezer.</li>
		</ol>
		</li>
		<li>Paclitaxel
		<ol>
			<li>Prepare a 1 mL solution of 1 mM paclitaxel diluted in DMSO. Pipet the solution into 10 &#956;L aliquots. Store the aliquots in a -20 &#176;C freezer.</li>
		</ol>
		</li>
		<li>Dimethyl sulfoxide (DMSO) 
		NOTE: The DMSO used in these experiments is pure.
		<ol>
			<li>Pipet 1 mL of pure DMSO into 10 &#956;L aliquots. Store the aliquots in a -20 &#176;C freezer.</li>
		</ol>
		</li>
		<li>Creatine phosphate
		<ol>
			<li>Prepare a 1 mL solution of 0.2 M creatine phosphate in ultrapure water. Pipet the solution into 10 &#956;L aliquots. Store the aliquots in a -20 &#176;C freezer.</li>
		</ol>
		</li>
		<li>Creatine phosphokinase
		<ol>
			<li>Prepare a 1 mL solution of 200 units&#183;L-1 creatine phosphokinase in ultrapure water. Pipet the solution into 10 &#956;L aliquots. Store the aliquots in a -20 &#176;C freezer.</li>
		</ol>
		</li>
		<li>Nickel (II) sulfate
		<ol>
			<li>Prepare 500 mL of a solution of 50 mM nickel (II) sulfate in ultrapure water. Store the solution at room temperature (~25 &#176;C).</li>
		</ol>
		</li>
		<li>Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (PEG-PPG-PEG) solution
		<ol>
			<li>Weigh out 2 mg of PEG-PPG-PEG (number average molecular weight: 14,600 g&#8729;mol-1)-NTA powder on weighing paper. PEG-PPG-PEG-NTA is the triblock copolymer functionalized with a nitrilotriacetic acid (NTA) group. Refer to the Table of Materials for more details about the PEG-PPG-PEG-NTA.</li>
			<li>Transfer the powder into a 1.5 mL microcentrifuge tube. Add 1 mL of the stock nickel (II) sulfate solution to the tube. Vortex until powder is dissolved and no visible clumps remain.</li>
			<li>Store for up to one month at room temperature.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Before starting an experiment
	<ol>
		<li>Fill a bucket with ice.</li>
		<li>Take one aliquot from each of the 13 reagents described in sections 1.1.1 to 1.1.13 above and add them to the bucket, thus keeping them on ice. Thaw each of the reagents before use.</li>
	</ol>
	</li>
	<li>Microtubule Preparation 
	NOTE: Microtubules were polymerized from a 20 &#956;g aliquot of dye-labeled lyophilized tubulin. The excitation wavelength of the dye is of 647 nm.
	<ol>
		<li>Preparation of the microtubule growth buffer
		<ol>
			<li>Pipet 21.8 &#956;L of BRB80 buffer into a small (0.6 mL) microcentrifuge tube. Add 1 &#956;L of the MgCl2 stock solution, 1 &#956;L of the GTP stock solution, and 1.2 &#956;L of the DMSO stock solution. 
			NOTE: Thus, the final concentrations of the reagents in the BRB80 buffer are 4 mM MgCl2, 1 mM GTP, and 5% (v/v) dimethyl sulfoxide.</li>
		</ol>
		</li>
		<li>Microtubule polymerization
		<ol>
			<li>Add 6.25 &#956;L of microtubule growth buffer directly into a 20 &#956;g aliquot of labeled lyophilized tubulin. Vortex the aliquot for 5 s at 30 rps.</li>
			<li>Cool the aliquot on ice for 5 min before incubating it at 37 &#176;C for 45 min and proceed to section 1.3.3.</li>
		</ol>
		</li>
		<li>Microtubule stabilization
		<ol>
			<li>Add 5 &#956;L of the aliquoted paclitaxel solution to 490 &#956;L of BRB80 buffer. Vortex the solution for 10 s at 30 rps.</li>
			<li>Once the 45 min of incubation for the microtubules are up, add 5 &#956;L of the polymerized microtubule solution to the BRB80/paclitaxel solution. 
			NOTE: This 100-fold diluted, stabilized, microtubule solution will hereafter be referred to as MT100. It can be used for up to 5 days, and it can be diluted to obtain the desired microtubule density for each experiment.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Motility solution
	<ol>
		<li>Enzymatic antifade, ATP regenerating system, and ATP
		<ol>
			<li>Add 9.0 &#956;L of the aliquoted casein solution to 291 &#956;L of BRB80 buffer.</li>
			<li>If the desired ATP concentration for the experiment is lower than 1 mM, pipet 83 &#956;L of the BRB80/casein solution into a new 0.6 mL microcentrifuge tube. Otherwise, pipet 85 &#956;L of that solution into a new 0.6 mL microcentrifuge tube.</li>
			<li>Add 1 &#956;L of D-glucose, 1 &#956;L of glucose oxidase, 1 &#956;L of catalase, and 1 &#956;L of DTT to that tube. 
			NOTE: These chemicals constitute an enzymatic antifade cocktail21 that will reduce photobleaching by removing dissolved oxygen and quenching reactive radicals. This reduces photobleaching and microtubule disintegration caused by the excitation illumination during fluorescence imaging22,23.</li>
			<li>For experiments in which the ATP concentration is chosen to be significantly lower than 1 mM, add the following reagents to the solution to create an ATP-regenerating system: 1 &#956;L of the aliquoted creatine phosphate solution and 1 &#956;L of the aliquoted creatine phosphokinase solution.</li>
			<li>Add 1 &#956;L of the stock ATP solution to the motility solution. Flick or vortex the aliquot to homogenously distribute the chemicals. 
			NOTE: Thus, the final concentration of chemicals in the solution are 10 &#956;M paclitaxel, 0.5 mg&#183;mL&#8722;1 casein, 20 mM D-glucose, 200 &#956;g&#183;mL&#8722;1 glucose oxidase, 8 &#956;g&#183;mL&#8722;1 catalase, 10 mM dithiothreitol, 2 mM creatine phosphate (if added), 2 units&#183;L&#8722;1 creatine phosphokinase (if added), and 1 mM ATP. This solution will hereafter be referred to as the motility solution.</li>
		</ol>
		</li>
		<li>Kinesin 
		NOTE: The stock kinesin solution used in these experiments is prepared by G. Bachand at the Center for Integrated Nanotechnologies at Sandia National Laboratories and made available under a user agreement (https://cint.lanl.gov/becoming-user/call-for-proposals.php). The buffer used here consists of 40 mM imidazole, 300 mM NaCl, 0.76 g&#183;L-1 EGTA, 37.2 mg&#183;L-1 EDTA, 50 g&#183;L-1 sucrose, 0.2 mM TCEP, and 50 &#956;M Mg-ATP. For this series of experiments, the rkin430eGFP kinesin construct was used. This is a kinesin consisting of the first 430 amino acids of rat kinesin heavy chain fused to eGFP and a C-terminal His-tag at the tail domain24. It was expressed in Escherichia coli and purified using a Ni&#8722;NTA column. The concentration of the GFP-kinesin stock solution was 1.8 &#177; 0.3 &#956;M as determined by UV/Vis spectrophotometry, using an extinction coefficient for GFP of 55 mM-1&#183;cm-1 at 489 nm25, and taking into account that kinesin is a dimer.
		<ol>
			<li>Determine the desired kinesin concentration or surface density for the experiment. For typical assays, this concentration is 20 nM. If the kinesin concentration needs to be diluted more than a hundred-fold, dilute it in a solution of BRB80 that contains 0.5 mg&#183;mL-1 of casein.</li>
			<li>Add 1 &#181;L of kinesin solution to the motility solution in order to get a final concentration of approximatively 20 nM.</li>
		</ol>
		</li>
		<li>Microtubules
		<ol>
			<li>Add 10 &#956;L of the MT100 solution prepared in section 1.3 to the motility solution. 
			NOTE: This motility solution can be used for up to 3 hours. After that time, the antifade system loses its effectiveness because the glucose in the solution is depleted by the enzymatic reaction.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Assembling the flow cells
<ol>
	<li>Washing the coverslips
	<ol>
		<li>For flow cells, use a large coverslip (dimension: 60 mm x 25 mm) and a small one (dimensions: 22 mm x 22 mm)19.</li>
		<li>Rinse all coverslips twice with ethanol and twice with ultrapure water. Sonicate the coverslips in ultrapure water for 5 min. Dry them in an oven at a temperature of 50&#8211;75 &#176;C.</li>
		<li>Using a UV/ozone cleaner (see Table of Materials and manufacturer&#8217;s instructions), treat one side of each coverslip for 15 min. Perform this step can at room temperature (~25 &#176;C) and in normal atmospheric condition (pressure of 1 atm).</li>
		<li>Carefully turn each coverslip to its other side (using tweezers) and UV/ozone treat that side as well.</li>
		<li>Sonicate the coverslips in ultrapure water again for 5 min before drying them again in the oven at a temperature of 50&#8211;75 &#176;C.</li>
	</ol>
	</li>
	<li>Treating the coverslips to enable PEG-PPG-PEG coating 
	NOTE: The dimethyldichlorosilane and the toluene used in this part of the protocol being highly toxic, perform the following steps under a fume hood, while taking the following precautions: wear two sets of protective gloves and a long-sleeved shirt under their lab coat. Tuck the edges of the gloves inside the sleeves of the shirt so that no skin from the forearms is directly exposed to the chemicals in case of a spillage. Wear protective glasses.
	<ol>
		<li>Dilute 25 mL of pure dimethyldichlorosilane in 475 mL of toluene. Immerse each coverslip in the dimethyldichlorosilane and toluene solution for 15 seconds.</li>
		<li>Wash the coverslips twice in toluene and three times in methanol. Dry the coverslips using pressurized nitrogen.</li>
	</ol>
	</li>
	<li>Assembling coverslips into flow cells
	<ol>
		<li>Once the coverslips are dry, cut a 2 cm x 2.5 cm piece of double-sided tape vertically into two 1 cm x 2.5 cm stripes.</li>
		<li>Put the large coverslip on a delicate task wiper and stick the tape stripes lengthwise along the edges of the coverslip to create a 1 cm x 2.5 cm area between the pieces of tape.</li>
		<li>Stick the small coverslip on top of the tape stripes to finish the flow cell assembly.</li>
	</ol>
	</li>
</ol>
3. Flowing the solutions into a flow cell
<ol>
	<li>Flow approximatively 20 &#956;L of the PEG-PPG-PEG solution into the assembled flow cell. The volume that is flown in the cell has to be large enough to fill the chamber. In the next steps, use the same volume when exchanging the solutions.</li>
	<li>Allow for the PEG-PPG-PEG solution to absorb on the surface for 5 minutes. Exchange the PEG-PPG-PEG solution with BRB80 buffer 3 times by flowing the buffer in. Flow the motility solution into the flow cell.</li>
	<li>Seal the edges of the flow cell with grease to prevent evaporation if the planned experiment is longer than an hour. 
	NOTE: The flow cell is now ready to be imaged.</li>
</ol>
4. Imaging a flow cell
<ol>
	<li>Perform the imaging using an objective-type total internal reflection fluorescence (TIRF) setup for both the microtubules and the kinesin motors (see Table of Materials). 
	NOTE: Here, we used a microscope with a 100x/1.49 numerical aperture objective lens, using two lasers, one with a wavelength of 642 nm and a maximum power of 140 mW, and another with wavelength of 488 nm and a maximum power of 150 mW.</li>
	<li>Place a drop of immersion oil on the objective.</li>
	<li>Place the flow cell on the microscope platform and bring the objective up until there is contact between the oil on the objective and the flow cell.</li>
	<li>Use the microscope&#8217;s interlock cover system to block all laser light from escaping.</li>
	<li>Turn on the laser and focus on the lower surface of the flow cell. The microtubules are fluorescently labelled and excited at a wavelength of 647 nm. They will be imaged using a 642 nm laser while a 488 nm laser is used for the GFP-kinesin motors.</li>
	<li>Record the images or videos of interest. Typically, the laser power is of about 30 mW and an exposure time of 50 ms for both laser channels. Images can be recorded for as long as there is motility in the flow cell. 
	NOTE: The laser illumination is harmful for the naked eye and can cause irreparable damage. Please make sure that the illuminated area is completely covered with an opaque lid.</li>
</ol>

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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Proteins in Food Processing (Second Edition). , 49-92 (2018).</li><li>Wettermark, G., Borglund, E., Brolin, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+regenerating+system+for+studies+of+phosphoryl+transfer+from+ATP.">A regenerating system for studies of phosphoryl transfer from ATP.</a> Analytical Biochemistry. 22, 211-218 (1968).</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> Journal of Cell Biology. 107, 1011-1024 (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, S540-S548 (2004).</li><li>Rogers, K. R., 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=KIF1D+is+a+fast+non-processive+kinesin+that+demonstrates+novel+K-loop-dependent+mechanochemistry.">KIF1D is a fast non-processive kinesin that demonstrates novel K-loop-dependent mechanochemistry.</a> EMBO Journal. 20, 5101-5113 (2001).</li><li>Patterson, G. H., Knobel, S. M., Sharif, W. D., Kain, S. R., Piston, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+green+fluorescent+protein+and+its+mutants+in+quantitative+fluorescence+microscopy.">Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy.</a> Biophysical Journal. 73, 2782-2790 (1997).</li></ol>

The authors have nothing to disclose.

In this work, we present an active nanoscale system which self assembles weakly-binding building blocks to construct its own track. As shown in Figure 1, gliding microtubules accumulate kinesin motors from solution and deposit them on the surface. The kinesin motors remain in the wake of the microtubule for a short period of time before returning to solution. Thus, in this experiment, kinesin motors alternate between 3 states:
(1) A microtubule single-bound state: this is when a kinesin first binds to a microtubule. It exists in equilibrium with state (2).
(2) A double-bound state: in this case, a microtubule single-bound kinesin also binds to the surface via its His-tag. This double-bound state allows for microtubule propulsion.
(3) A single surface-bound state: a double-bound kinesin that has walked off the end of the microtubule and has not yet desorbed from the surface is in this state. These motors can be observed in Figure 1 (combined and green channels): they extend behind the tail of the microtubule for several micrometers and form its diminishing trail.
The most critical step of this protocol is the formation of the hydrophobic surface on the slide. Not only does it use dangerous chemicals, but it also allows the PEG-PPG-PEG functionalized with the NTA group to coat the surface, which then allows the kinesin to reversibly bind to the surface. Another important step is sealing the flow cell with grease. This allows for prolonged imaging without the liquid in the flow cell evaporating.
The primary modifications to this technique consist of changing microtubule concentration, kinesin concentration, and ATP concentration. Changing microtubule concentration will change the number of microtubules gliding on the surface. Changing kinesin concentration will change the number of kinesin molecules that can bind to the microtubule. However, increasing the kinesin concentration above the amounts already defined in this experiment could increase background fluorescence, making it more difficult to see the kinesin trails left behind gliding microtubules. Meanwhile, lowering ATP concentrations below 10 &#181;M will significantly decrease microtubule gliding velocity. If this effect is desired, it is necessary to utilize an ATP regenerating system consisting of creatine phosphatase and phosphokinase.
A possible limitation of this technique is that, due to the large active kinesin content of the system, the ATP can be rapidly consumed, and experiments may last less than an hour in certain conditions. This would for example be the case if one used a twofold higher kinesin concentration and five-fold higher microtubule concentration than what is presented in this protocol.
In our previous work18, we studied the spatial distribution of kinesin motors along the microtubules, proving that gliding microtubules accumulate kinesin motors from solution, resulting in an increase of the density of motors along the length of the microtubule. We also found that the microtubules&#39; gliding stability demonstrated a nonlinear dependence on the solution kinesin concentration and microtubule velocity.
The presented protocol paves the way for a more efficient use of protein motors in nanoscale engineered systems and for further investigation in the design of active nanosystems that are in dynamic equilibrium. Furthermore, the dynamic nature of this system allows it to serve as a model system for studying self-healing and dynamic replacement of molecular components, closing part of the gap between engineered and natural structures.

The interactions governing the behavior of active nanosystems have always been characterized by long-lived, nearly irreversible bonds1,2,3,4,5,6,7,8. A well-studied example of this is the microtubule-kinesin system, where gliding microtubules are propelled by irreversibly surface-bound kinesin motors1,2,3,4,5. Systems in which the components are reversibly connected to one another have been studied theoretically9,10 and achieved at the macroscale11,12, but scaling these systems down to the nanoscale has been challenging. One of the major reasons for this is that breaking and reforming bonds between components often requires a large change in the environmental conditions. Even though such changes have been implemented in the past13,14,15, they would rely on modifying the system itself rather than adapting it to its environment. Designing molecular-scale systems in which components continually assemble and reorganize into structures without disturbing the overall environment in which the experiments take place will open the door to the exploration of a wide range of dynamic behaviors16,17.
Here, we describe and demonstrate the detailed protocol for creating a dynamically assembling and disassembling system functioning at the nanoscale. The system and its general behavior has been introduced earlier18: microtubule filaments are propelled by tracks of reversibly surface-bound kinesin-1 motors. These kinesin motor proteins are recruited from the solution to help propel microtubules forward, before desorbing again shortly afterwards. Once back in solution, they can be recruited again to propel a new microtubule. In the past13,14,15, the breaking and reforming of bonds required environmental modifications; in contrast, the environment of our flow cell remains unchanged while the kinesin motors interact with the surface.
This protocol will help interested researchers to (1) visualize all the steps of the protocol, and (2) assist with troubleshooting this type of assay. It has been derived from the procedures described in Howard et al. 199319.

<table><tbody><tr><td>488 nm laser</td><td>Omicron Laserage</td><td>LuxX 488-150</td><td></td></tr><tr><td>642 nm laser</td><td>Omicron Laserage</td><td>LuxX&amp;nbsp;642</td><td></td></tr><tr><td>Casein</td><td>Sigma</td><td>C7078-500G</td><td></td></tr><tr><td>Catalase from bovine liver</td><td>Sigma</td><td>C40-500MG</td><td></td></tr><tr><td>Creatine Phosphate</td><td>Sigma</td><td>P-7936</td><td></td></tr><tr><td>Creatine Phosphokinase</td><td>Sigma</td><td>C3755-500UN</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma</td><td>G2133-50KU</td><td></td></tr><tr><td>Dichlorodimethylsilane solution</td><td>Sigma</td><td>40140-25ML</td><td>Toxic</td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma</td><td>34869-100ML</td><td></td></tr><tr><td>Dithiothreitol</td><td>Sigma</td><td>D0632-5G</td><td>Toxic</td></tr><tr><td>Eclipse TI</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>eGFP rkin430</td><td>Provided by George Bachand</td><td></td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E4378-25G</td><td></td></tr><tr><td>Falcon 50 mL Conical Centrifuge Tubes</td><td>Fisher Scientific</td><td>14-959-49A</td><td></td></tr><tr><td>Glucose Oxidase</td><td>Sigma</td><td>G0543-10KU</td><td></td></tr><tr><td>Guanosine Triphosphate</td><td>Sigma</td><td>G8877-10MG</td><td></td></tr><tr><td>Kimwipes Delicate Task Wipers</td><td>Sigma Pharmaceuticals</td><td>8089</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Sigma</td><td>M1028-100ML</td><td></td></tr><tr><td>Methanol</td><td>Fisher Chemical</td><td>A412</td><td>Toxic</td></tr><tr><td>Milli-Q Water Purification System</td><td>Millipore Corporation</td><td></td><td></td></tr><tr><td>Nickel Sulfate</td><td>Sigma</td><td>656895-50G</td><td></td></tr><tr><td>Paclitaxel</td><td>Sigma</td><td>T1912-5MG</td><td></td></tr><tr><td>PIPES</td><td>Sigma</td><td>P-6757</td><td></td></tr><tr><td>Pluronic F108-NTA</td><td>Provided by Jennifer Neff and AllVivo Vascular</td><td></td><td>PEG-PPG-PEG-NTA</td></tr><tr><td>Pluronic&amp;nbsp;F-108</td><td>Sigma</td><td>542342-250G</td><td>PEG-PPG-PEG</td></tr><tr><td>Thermo Scientific Snap Cap Low Retention Microcentrifuge Tubes</td><td>Fisher Scientific</td><td>21-403-190</td><td></td></tr><tr><td>Toluene</td><td>Fisher Chemical</td><td>T324</td><td>Toxic</td></tr><tr><td>Tubulin, HiLyte647-labeled</td><td>Cytoskeleton, Inc.</td><td>TL670M</td><td></td></tr><tr><td>UV Ozone Procleaner</td><td>BioForce Nanosciences</td><td>PC440</td><td></td></tr><tr><td>Whatman&amp;nbsp;Puradisc syringe filters</td><td>Sigma</td><td>WHA67840402</td><td></td></tr><tr><td>Zyla 4.2 sCMOS Camera</td><td>Andor Technology</td><td>sCMOS 4.2</td><td></td></tr></tbody></table>

assembling molecular shuttles powered by reversibly attached kinesins

This protocol describes how to create kinesin-powered molecular shuttles with a weak and reversible attachment of the kinesins to the surface. In contrast to previous protocols, in this system, microtubules recruit kinesin motor proteins from solution and place them on a surface. The kinesins will, in turn, facilitate the gliding of the microtubules along the surface before desorbing back into the bulk solution, thus being available to be recruited again. This continuous assembly and disassembly leads to striking dynamic behavior in the system, such as the formation of temporary kinesin trails by gliding microtubules.
Several experimental methods will be described throughout this experiment: UV-Vis spectrophotometry will be used to determine the concentration of stock solutions of reagents, coverslips will first be ozone and ultraviolet (UV) treated and then silanized before being mounted into flow cells, and total internal reflection fluorescence (TIRF) microscopy will be used to simultaneously image kinesin motors and microtubule filaments.

In these experiments, we used a 1,000 times dilution of the microtubules prepared in section 1.3.2. The kinesin concentration was 20 nM, and the ATP concentration was 1 mM. Imaging was performed using TIRF microscopy. Gliding microtubules were separately imaged from the kinesin motors: microtubules were visible upon excitation with a 647 nm laser (Figure 1, red), and the GFP-kinesin was visible when excited with a 488 nm laser (Figure 1, green). The time between excitation with the red and green light was less than 1 s. The time between frames was 10 s. Microtubules displayed stable gliding. The average microtubule gliding velocity was approximatively 800 nm/s. The microtubule surface density was 400 mm-2. Tracks of kinesin (green) appeared to extend beyond the trailing end of the microtubules (red) for several micrometers.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59068/59068fig1.jpg" /> 
Figure 1: Gliding microtubules propelled by weakly surface-bound kinesin motors. As microtubules move forward, they accumulate kinesin motors from solution. These motors alternate between two states: single-bound to the microtubule, and double-bound to both the microtubule and the surface. When a double bound motor reaches the end of the microtubule, the motor is left behind and slowly desorbs from the surface with an off rate of approximately 0.1 s-1. As a result, trails of kinesin motors remain behind the microtubules. Top row: red (microtubule) channel. Middle row: green (kinesin) channel. Bottom row: combined red (microtubule) and green (GFP-kinesin) channel. Scale bar: 20 &#181;m. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59068/59068fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Assembling Molecular Shuttles Powered by Reversibly Attached Kinesins at JoVE.com

Assembling Molecular Shuttles Powered by Reversibly Attached Kinesins

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.

This protocol describes how to create kinesin-powered molecular shuttles with a weak and reversible attachment of the kinesins to the surface. In contrast ...

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Universidad de Cartagena UDEC

Research

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Engineering

6.8K Views.  Columbia University. 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.

Video: Assembling Molecular Shuttles Powered by Reversibly Attached Kinesins

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.

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 ...

Biochemistry

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

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.

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of ...

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 ...

Biology

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

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.

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

Self-Assembly of Microtubule Tactoids

The cytoskeleton is responsible for major internal organization and re-organization within the cell, all without a manager to direct the changes. This is especially the case during mitosis or meiosis, where the microtubules form the spindle during cell division. The spindle is the machinery used to segregate genetic material during cell division. Toward creating self-organized spindles in vitro, we recently developed a technique to reconstitute microtubules into spindle-like assemblies with a minimal set of microtubule-associated proteins and crowding agents. Specifically, MAP65 was used, which is an antiparallel microtubule crosslinker from plants, a homolog of Ase1 from yeast and PRC1 from mammalian organisms. This crosslinker self-organizes microtubules into long, thin, spindle-like microtubule self-organized assemblies. These assemblies are also similar to liquid crystal tactoids, and microtubules could be used as mesoscale mesogens. Here, protocols are presented for creating these microtubule tactoids, as well as for characterizing the shape of the assemblies using fluorescence microscopy and the mobility of the constituents using fluorescence recovery after photobleaching.

This article presents a protocol for the formation of microtubule assemblies in the shape of tactoids using MAP65, a plant-based microtubule crosslinker, and PEG as a crowding agent.

The cytoskeleton is responsible for major internal organization and re-organization within the cell, all without a manager to direct the changes. This is ...

Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are thought to play a vital role in the microtubule-dependent chromosome motions during cell division, but the relevant proteins and their exact roles are ill-defined. Thus, there is a growing need to develop assays with which to study such motility in vitro using purified components and defined biochemical milieu. Microtubules, however, are inherently unstable polymers; their switching between growth and shortening is stochastic and difficult to control. The protocols we describe here take advantage of the segmented microtubules that are made with the photoablatable stabilizing caps. Depolymerization of such segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting studies of motility at the disassembling microtubule ends. This technique can be used to carry out a quantitative analysis of the number of molecules in the fluorescently-labeled protein complexes, which move processively with dynamic microtubule ends. To optimize a signal-to-noise ratio in this and other quantitative fluorescent assays, coverslips should be treated to reduce nonspecific absorption of soluble fluorescently-labeled proteins. Detailed protocols are provided to take into account the unevenness of fluorescent illumination, and determine the intensity of a single fluorophore using equidistant Gaussian fit. Finally, we describe the use of segmented microtubules to study microtubule-dependent motions of the protein-coated microbeads, providing insights into the ability of different motor and nonmotor proteins to couple microtubule depolymerization to processive cargo motion.

Microtubules are inherently unstable polymers, and their switching between growth and shortening is stochastic and difficult to control. Here we describe protocols using segmented microtubules with photoablatable stabilizing caps. Depolymerization of segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting analysis of motions with the disassembling microtubule ends.

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are ...

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 ...

Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and cross-linkers. Growing microtubules can generate pushing forces, while depolymerizing microtubules can convert the energy from microtubule shrinkage into pulling forces, when attached, for example, to cortical dynein or chromosomes. In addition, motor proteins and diffusible cross-linkers within the spindle contribute to spindle architecture by connecting and sliding anti-parallel microtubules. In vivo, it has proven difficult to unravel the relative contribution of individual players to the overall balance of forces. Here we present the methods that we recently developed in our efforts to reconstitute basic mitotic spindles bottom-up in vitro. Using microfluidic techniques, centrosomes and tubulin are encapsulated in water-in-oil emulsion droplets, leading to the formation of geometrically confined (double) microtubule asters. By additionally introducing cortically anchored dynein, plus-end directed microtubule motors and diffusible cross-linkers, this system is used to reconstitute spindle-like structures. The methods presented here provide a starting point for reconstitution of more complete mitotic spindles, allowing for a detailed study of the contribution of each individual component, and for obtaining an integrated quantitative view of the force-balance within the mitotic spindle.

The assembly and positioning of the mitotic spindle depend on the combined forces generated by microtubule dynamics, motor proteins and cross-linkers. Here we present our recently developed methods in which the geometrical confinement of spherical emulsion droplets is used for the bottom-up reconstitution of basic mitotic spindles.

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and ...

Directly Measuring Forces Within Reconstituted Active Microtubule Bundles

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as specialized arrays during mitosis to regulate chromosome segregation. Proteins that interact with microtubules include motors such as kinesins and dynein, which can generate active forces and directional motion, as well as non-motor proteins that crosslink filaments into higher-order networks or regulate filament dynamics. To date, biophysical studies of microtubule-associated proteins have overwhelmingly focused on the role of single motor proteins needed for vesicle transport, and significant progress has been made in elucidating the force-generating properties and mechanochemical regulation of kinesins and dyneins. However, for processes in which microtubules act both as cargo and track, such as during filament sliding within the mitotic spindle, much less is understood about the biophysical regulation of ensembles of the crosslinking proteins involved. Here, we detail our methodology for directly probing force generation and response within crosslinked microtubule minimal networks reconstituted from purified microtubules and mitotic proteins. Microtubule pairs are crosslinked by proteins of interest, one microtubule is immobilized to a microscope coverslip, and the second microtubule is manipulated by an optical trap. Simultaneous total internal reflection fluorescence microscopy allows for multichannel visualization of all the components of this microtubule network as the filaments slide apart to generate force. We also demonstrate how these techniques can be used to probe pushing forces exerted by kinesin-5 ensembles and how viscous braking forces arise between sliding microtubule pairs crosslinked by the mitotic MAP PRC1. These assays provide insights into the mechanisms of spindle assembly and function and can be more broadly adapted to study dense microtubule network mechanics in diverse contexts, such as the axon and dendrites of neurons and polar epithelial cells.

Here, we present a protocol for reconstituting microtubule bundles in vitro and directly quantifying the forces exerted within them using simultaneous optical trapping and total internal reflection fluorescence microscopy. This assay allows for nanoscale-level measurement of the forces and displacements generated by protein ensembles within active microtubule networks.

Microtubule networks are employed in cells to accomplish a wide range of tasks, ranging from acting as tracks for vesicle transport to working as ...

Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and Mechanics

The composite cytoskeleton, comprising interacting networks of semiflexible actin filaments and rigid microtubules, restructures and generates forces using motor proteins such as myosin II and kinesin to drive key processes such as migration, cytokinesis, adhesion, and mechanosensing. While actin-microtubule interactions are key to the cytoskeleton's versatility and adaptability, an understanding of their interplay with myosin and kinesin activity is still nascent. This work describes how to engineer tunable three-dimensional composite networks of co-entangled actin filaments and microtubules that undergo active restructuring and ballistic motion, driven by myosin II and kinesin motors, and are tuned by the relative concentrations of actin, microtubules, motor proteins, and passive crosslinkers. Protocols for fluorescence labeling of the microtubules and actin filaments to most effectively visualize composite restructuring and motion using multi-spectral confocal imaging are also detailed. Finally, the results of data analysis methods that can be used to quantitatively characterize non-equilibrium structure, dynamics, and mechanics are presented. Recreating and investigating this tunable biomimetic platform provides valuable insight into how coupled motor activity, composite mechanics, and filament dynamics can lead to myriad cellular processes from mitosis to polarization to mechano-sensation.

This paper presents protocols for engineering and characterizing tunable three-dimensional composite networks of co-entangled actin filaments and microtubules. Composites undergo active restructuring and ballistic motion, driven by myosin II and kinesin motors, and are tuned by the relative concentrations of actin, microtubules, motor proteins, and passive crosslinkers.

The composite cytoskeleton, comprising interacting networks of semiflexible actin filaments and rigid microtubules, restructures and generates forces ...

Assembling Molecular Shuttles Powered by Reversibly Attached Kinesins

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Directly Measuring Forces Within Reconstituted Active Microtubule Bundles

Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and Mechanics