We acknowledge Maya Hendija and Dr. Jonathan Michel for assistance with data analysis, and Dr. Janet Sheung, Dr. Moumita Das, and Dr. Michael Rust for helpful discussions and guidance. This research was supported by a William M. Keck Foundation Research Grant and NSF DMREF Award (DMR 2119663) awarded to RMRA and JLR and National Institutes of Health R15 Grants (R15GM123420, 2R15GM123420-02) awarded to RMR-A and RJM.

1. Prepare silanized coverslips and microscope slides to prevent adsorption of proteins to chamber surfaces
NOTE: This is a 2-day process. Silanized slides may be prepared up to 1 month in advance of use.
<ol>
	<li>Place no. 1 coverslips (24 mm x 24 mm) and microscope slides (1 in x 3 in) in a designated rack that will fit in the plasma cleaner. Place rack in plasma cleaner and run for 20 min.</li>
	<li>Transfer coverslips and slides to a new rack designated only for use with silane and place rack in glass container to clean the glasses as described below.
	<ol>
		<li>Immerse coverslips and slides in 100% acetone for 1 h. Immerse coverslips and slides in 100% ethanol for 10 min.</li>
		<li>Immerse coverslips and slides in deionized water (DI) for 5 min. Repeat the cleaning steps two more times.</li>
		<li>Immerse coverslips and slides in freshly prepared 0.1 M KOH for 15 min. Immerse coverslips and slides in fresh DI for 5 min. Repeat this step two more times.</li>
	</ol>
	</li>
	<li>Air dry coverslips and slides for 10 min. Treat cleaned coverslips and slides with silane to produce hydrophobic surfaces as described below. 
	NOTE: Complete the following steps in a fume hood.
	<ol>
		<li>Immerse&#160;dried coverslips and slides in 2% silane (dissolved in toluene) for 5 min. Use a funnel to pour silane back into its designated bottle to reuse up to five times.</li>
		<li>Immerse coverslips and slides in 100% ethanol for 5 min. Replace ethanol with fresh ethanol. Immerse coverslips and slides for 5 min.</li>
		<li>Immerse coverslips and slides in fresh DI for 5 min. Repeat the ethanol and DI wash step two more times using fresh ethanol and DI each time. Air dry coverslips and slides for 10 min.</li>
	</ol>
	</li>
</ol>
2. Preparing active actin-microtubule composite driven by myosin mini-filaments
<ol>
	<li>Remove inactive myosin via actin filament binding and perform pull-down via ultracentrifugation as described below.
	<ol>
		<li>Polymerize actin into filaments. Using a precision micropipette and sterile pipet tips, combine in a microcentrifuge tube: 1.87 &#181;L of DI, 1.3 &#181;L of 10x G-buffer, 1.3 &#181;L of 10x F-buffer, 1.63 &#181;L of 4 M KCl, 4.53 &#181;L of actin (47.6 &#181;M), and 1.08 &#181;L of 100 &#181;M phalloidin. 
		NOTE: To ensure sufficient polymerization, the actin concentration and actin:phalloidin molar ratio should be 18.4 &#181;M and 2:1, respectively.</li>
		<li>Gently pipet the solution up and down to mix and then set on ice in the dark for &#8805;1 h. Cool ultracentrifuge to 4 &#176;C. Remove myosin aliquot from -80 &#176;C and put on ice. 
		NOTE: Complete step 2.2 at this point while actin is polymerizing.</li>
		<li>After &#8805;1 h of actin polymerization, add 1.3 &#181;L of 10 mM ATP and 2 &#181;L of 19 &#181;M myosin to the polymerized actin. 
		NOTE: The actin:myosin molar ratio should be &#62;5 to ensure sufficient removal of inactive myosin motors (i.e., dead heads).</li>
		<li>Gently pipet the solution up and down to mix. Transfer into an ultracentrifuge grade tube. 
		&#8203;Centrifuge at 4 &#176;C and 121,968 x g for 30 min.</li>
	</ol>
	</li>
	<li>Prepare co-entangled composite network of actin filaments and microtubules as described below. 
	NOTE: Begin 30 min before myosin spin-down (step 2.1.4).
	<ol>
		<li>Set a heat block to 37 &#176;C. Use a precision micropipette and sterile pipet tips to add the following to a microcentrifuge tube: 13.9 &#181;L of PEM, 3 &#181;L of 1% Tween20, 1.55 &#181;L of 47.6 &#181;M actin, 0.36 &#181;L of 34.8 &#181;M R-actin, 0.3 &#181;L of 250 mM ATP, 0.87 &#181;L of 100 &#181;M phalloidin, 1.91 &#181;L of 5-488-tubulin, 0.3 &#181;L of 100 mM GTP, and 0.75 &#181;L of 200 &#181;M Taxol, to a total volume of 23 &#181;L. 
		NOTE: The concentrations of actin and tubulin listed are for a composite with 2.9 &#181;M actin and 2.9 &#181;M tubulin. Total protein concentration is c = cA + cT&#160;= 5.8 &#956;M&#160;and molar actin fraction is cA/(cA + cT) = &#934;A = 0.5. See step 2.5 to adjust these values.</li>
		<li>Gently pipet the solution up and down to mix and place on a 37 &#176;C heat block protected from light for 1 h.</li>
	</ol>
	</li>
	<li>Prepare sample chambers for confocal imaging experiments as described below. 
	NOTE: Complete steps 2.1.4 and 2.2.2 during waiting periods.
	<ol>
		<li>Place two silanized slides side-by-side on a hot plate (turned off), lay two strips of thermoplastic sealing film across the slides ~3 mm apart, and place two silanized coverslips over the thermoplastic sealing film to form a sample chamber.</li>
		<li>Turn the hot plate on low setting until coverslips firmly bind to slides with melted thermoplastic sealing film (~1-2 min). Press down with even pressure to ensure bonding while maintaining ~100 &#181;m spacing between the two surfaces.</li>
		<li>Remove chambers and turn off hot plate. Label chambers with (+) and (-). The (+) chamber will be for the active sample (with myosin) and the (-) chamber will be the control (no myosin). Ensure each chamber can accommodate &#8804;10 &#181;L of fluid.</li>
	</ol>
	</li>
	<li>Prepare samples to image as described below. 
	NOTE: It is important to complete this step immediately after steps 2.1 and 2.2 are completed.
	<ol>
		<li>Carefully remove the myosin-actin sample from the ultracentrifuge (step 2.1.4) and immediately pipet up the top 7.5 &#181;L of the supernatant and transfer to a new microcentrifuge tube.</li>
		<li>Remove the actin-microtubule sample from the heat block and gently mix in 1.5 &#181;L of 10x D-Glucose, 1.5 &#181;L of 10x GOC, and 1.5 &#181;L of 1 mM blebbistatin. Divide the solution into two 13.7 &#181;L aliquots and label as (+) and (-).</li>
		<li>Mix in 1.28 &#181;L of the supernatant from step 2.4.1 to (+) aliquot. Mix in 1.28 &#181;L of DI to the (-) aliquot. Slowly flow each solution into the corresponding chamber (step 2.3) via capillary action. Be careful not to introduce air bubbles into the channel.</li>
		<li>Seal the two open ends of each channel with fast-drying epoxy or UV glue. Ensure the adhesive is completely dry before placing on the microscope. Image immediately as described in step 3. 
		NOTE: UV glue is advantageous&#160;because it cures nearly instantly upon UV exposure. However, because blebbistatin is UV sensitive, it is important to only locally illuminate the glue (at the edges of the sample chamber) using a small UV wand to avoid deactivating the blebbistatin.</li>
	</ol>
	</li>
	<li>Optional: vary the protein concentrations to tune the dynamics and structure of the composites. 
	NOTE: The following steps are suggested alterations to the steps above to vary the concentrations of actin, microtubules, and myosin if desired.
	<ol>
		<li>Follow the steps described above except for the following modifications in steps 2.2.1 and 2.4.3.</li>
		<li>To vary the concentrations of actin and microtubules, thereby adjusting c and &#934;A, increase or decrease the volume of actin, R-actin, and 5-488-tubulin used in step 2.2.1, as desired26. When varying the actin concentration, adjust R-actin and phalloidin molar concentrations proportionally to maintain the same molar ratios with actin. Adjust the volume of PEM such that the final volume of the mixture remains 23 &#181;L. All other component volumes and concentrations remain the same.</li>
		<li>To vary the myosin concentration, adjust the volume of myosin added to the (+) aliquot in step 2.4.3 as desired. Adjust the DI volume added to the (-) aliquot accordingly. Adjust the PEM volume in Step 2.2.1 to account for the increase or decrease in myosin (+) and DI (-) volume, ensuring the final volume of each sample ((+) and (-)) is 14.98&#160;&#956;L.</li>
	</ol>
	</li>
</ol>
3. Imaging and characterization of active composites using confocal microscopy
<ol>
	<li>To image actomyosin-microtubule composites prepared in step 2, use a laser scanning confocal microscope (LSCM), or similar microscope, with a 60x 1.4 NA oil-immersion objective. To simultaneously visualize actin filaments and microtubules in separate fluorescence channels, use a 561 nm laser with 565/591 nm excitation/emission filters and a 488 nm laser with 488/525 nm excitation/emission filters.</li>
	<li>Place the sample chamber on the microscope such that the control channel is positioned directly over the objective. Make sure there is an oil interface between the objective and the coverslip.</li>
	<li>Use the stage controls to bring the control composite into focus, then find both surfaces of the sample chamber. Move the z position to the center of the sample chamber. Check for the presence of clear filamentous networks as shown in Figure 2.</li>
	<li>Still visualizing the control chamber, adjust the intensity of each laser to allow for simultaneous visualization of actin filaments and microtubules. Maintain the lowest laser intensity possible to prevent photobleaching (more prevalent in the actin channel) and bleed through (typically from the microtubules into the actin channel).</li>
	<li>To characterize the inactive control sample, collect three time-series (videos) of 256 x 256 square-pixel (213 &#181;m x 213 &#181;m) images at 2.65 fps for a total of &#8805;1000 frames. Collect each time-series in a different region of the sample chamber separated by &#8805;500 &#181;m. Ensure that there is minimal detectable motion and no flow or restructuring.</li>
	<li>Shutter off the 488 nm laser and use the stage controls to move to the (+) chamber.</li>
	<li>Using the 568 nm laser, visualize the microtubules in the (+) channel to ensure proper network formation (Figure 2) and identify the axial center of the sample chamber (which may be different to the center z-position of the control chamber).</li>
	<li>Turn on the 488 nm laser and repeat step 3.5 above with the following modifications. Collect time-series for up to 45 min, stopping acquisition when the sample either moves out of the field of view, ruptures, or photobleaches. Record 5-10 time-series and keep track of the time at which each time-series begins relative to the start of the first time-series.</li>
	<li>Analyze data using DDM, SIA, and PIV as described in Figure 3, Figure 4, Figure 5, and previously17,48,50,51. 
	&#8203;NOTE: The 488 nm laser locally activates myosin ATPase activity by de-activating blebbistatin, so it should only be turned on at the start of&#160;data acquisition such that t = 0 is at the start of the time-series. These acquisition parameters are optimized for differential dynamic microscopy (DDM) analysis as done previously26.</li>
</ol>
4. Preparation of active actin-microtubule composites driven by kinesin motors
NOTE: The following steps create actin-microtubule composites that are driven out-of-equilibrium by kinesin motors or a combination of kinesin and myosin50.
<ol>
	<li>Prepare kinesin and myosin motors as described below.
	<ol>
		<li>If incorporating myosin, follow step 2.1.</li>
		<li>To form kinesin motor clusters that bind and exert forces between pairs of microtubules, use a micropipette and sterile pipet tips to add the following to a sterile 1.5 mL microcentrifuge tube:1.16 &#181;L PEM, 2.74 &#181;L 8.87 &#181;M kinesin dimers, 7.29 &#181;L 83.3 &#181;M NeutrAvidin, 0.81 &#181;L 2mM DTT . Mix gently by pipetting the solution up and down and incubate protected from light (use a black microcentrifuge tube or wrap in foil) for 30 min at 4 &#176;C. 
		NOTE: The molar ratio of kinesin dimers to NA is 1:25.</li>
	</ol>
	</li>
	<li>Follow step 2.3 to prepare sample chambers and make three chambers instead of two. Carry out this step during kinesin incubation (step 4.1.2) and myosin ultracentrifugation (step 4.1.1).</li>
	<li>Prepare co-entangled composite network of actin filaments and microtubules.
	<ol>
		<li>Set heat block to 37 &#176;C. Use a micropipette and sterile pipet tips to add the following to a sterile 1.5 mL microcentrifuge tube: 3.21 &#181;L PEM, 4.5 &#181;L of 1% Tween20, 2.18 &#181;L of 47.6 &#181;M actin, 3.46 &#181;L of 5-R-tubulin, 4.5&#160;&#181;L of 100 mM ATP, 4.5 &#181;L of 10 mM GTP, 1.13 &#181;L of 200 &#181;M Taxol, and 1.57 &#181;L of 20 &#181;M 488-phalloidin. Ensure the total volume is 25 &#181;L.</li>
		<li>Gently pipet the solution up and down to mix and place on the 37 &#176;C heat block protected from light for 1 h. Remove the tube from the heat block and use a micropipette to gently mix in 0.84 &#181;L of 100&#160;&#956;M phalloidin. Incubate for 5-10 min at room temperature, protected from light. 
		NOTE: Adding phalloidin at this step, rather than in step 4.3.1, improves the fluorescence labeling of actin filaments, as 488-phalloidin does not have to compete with unlabeled phalloidin for actin binding sites.</li>
	</ol>
	</li>
	<li>Prepare active composites for confocal imaging.
	<ol>
		<li>Add 1.13 &#181;L of 200 &#956;M blebbistatin, 1.35 &#181;L of 10x Glu, and 1.35 &#181;L of 10x GOC to the solution from step 4.3.2 and mix gently by pipetting up and down. Divide the solution into three 10 &#181;L aliquots and label as (K), (K+M), and (-).</li>
		<li>Mix in 2.54 &#181;L of myosin from step 2.1.4 to the (K+M) aliquot. Mix in 2.54 &#181;L PEM to (K) and (-) aliquots.</li>
		<li>Use a micropipette and sterile pipet tips to add 2.5 &#181;L of kinesin clusters from step 4.1.2 to (K) and (K+M) aliquots. Pipet up and down to mix. Mix in 2.5 &#181;L PEM to (-) using the same technique. 
		NOTE: The concentrations of actin and tubulin listed are for a composite with 2.32 &#181;M actin and 3.48 &#181;M tubulin. Total protein concentration is c = cA&#160;+ cT&#160;= 5.8&#160;&#956;M&#160;and molar actin fraction is cA/(cA&#160;+ cT) =&#160;&#934;A&#160;= 0.4. Kinesin and myosin concentrations are 0.35 &#181;M and 0.47 &#181;M, respectively. See step 2.5 for general guidelines to adjust cA, cT, c, and &#934;A.</li>
		<li>Using a micropipette, slowly flow each solution into the corresponding channel of the prepared sample chambers (step 4.2) via capillary action. Push down very slowly and gently on the pipet so as to not introduce air bubbles into the channel.</li>
		<li>Seal the two open ends of each channel with fast-drying epoxy or UV-curable glue. Ensure the adhesive is completely dry before placing on the microscope. 
		NOTE: It is important that this step is done quickly to minimize the time that the kinesin is acting without being monitored. For this reason, epoxy that cures in 1 min (rather than 5 or 10 min) is recommended. UV-curable glue is advantageous in this regard because it cures nearly instantly upon UV exposure.&#160;</li>
	</ol>
	</li>
	<li>Image prepared samples immediately, following step 3, except for the following important modifications. Because kinesin is not controlled by light-activation, it starts to work immediately after step 4.4.3, so mark this time as t = 0. To image the composite as close to the initial inactive state (t = 0) as possible, image the (K) and (K+M) channels first and note the time elapsed between step 4.4.3 and the beginning of data acquisition (step 3.8). In practice, this elapsed time is ~5 min.</li>
</ol>
5. Incorporating passive crosslinkers into active composites
NOTE: These steps describe how to use biotinylated actin and tubulin subunits and NeutrAvidin (NA) to passively crosslink actin to actin (A-A) or microtubules to microtubules (M-M) in the active composites described in step 4.
<ol>
	<li>Prepare A-A or M-M crosslinker complexes with biotinylated proteins (biotin-actin or biotin-tubulin), NA, and biotin at a ratio of 2:2:1 biotin-actin/tubulin:biotin:NA. Start this process before Step 4 .
	<ol>
		<li>For A-A crosslinkers, use a micropipette and sterile pipet tips to add 2 &#181;L of 11.6 &#181;M biotin-actin, 1.39 &#181;L of 8.33 &#181;M NA, 2.27 &#181;L of 1.02 &#181;M biotin, and 4.34 &#181;L of PEM to a microcentrifuge tube. Mix gently by pipetting up and down.</li>
		<li>For M-M crosslinkers, use a micropipette and sterile pipet tips to add 1.86 &#181;L of 4.55 &#181;M biotin-tubulin, 1.11 &#181;L of 8.33 &#181;M NA, 1.82 &#181;L of 1.02 &#181;M biotin, and 5.21 &#181;L of PEM to a microcentrifuge tube. Mix gently by pipetting up and down.</li>
		<li>Wrap the tube(s) from step 5.1.1 and/or 5.1.2 in thermoplastic sealing film to create a water-tight seal. Place in a flotation raft in a temperature-controlled sonicator bath set to 4 &#176;C.</li>
		<li>Sonicate for 90 min at 4 &#176;C. In practice it is best to put the sonicator in a cold room and add ice packs to the sonication bath to maintain low temperature.</li>
	</ol>
	</li>
	<li>To incorporate crosslinker complexes into samples for imaging, follow step 4.3, modifying step 4.3.1 as described below for A-A crosslinking (step 5.2.1) or M-M crosslinking (step 5.2.2).
	<ol>
		<li>For A-A crosslinking, combine the following in a microcentrifuge tube: 1.94&#160;&#181;L of PEM, 4.50 &#181;L of 1% Tween20, 2.18 &#181;L of 47.6 &#181;M actin, 3.46 &#181;L of 45.5 &#181;M 5-R-tubulin, 1.13 &#181;L of A-A crosslinkers (step 5.1.1), 4.50 &#181;L of 100&#160;mM ATP, 4.50 &#181;L of 10 mM GTP, 1.13 &#181;L of 200 &#181;M Taxol, and 1.57 &#181;L of 20 &#181;M 488-phalloidin. Ensure the total volume is 25 &#181;L.</li>
		<li>For M-M crosslinking, combine the following in a microcentrifuge tube: 1.97&#160;&#181;L of PEM, 4.50 &#181;L of 1% Tween20, 2.18 &#181;L of 47.6 &#181;M actin, 3.76 &#181;L of 45.5 &#181;M 5-R-tubulin, 1.13 &#181;L of 1:4 dilution of M-M crosslinkers (step 5.1.2), 4.50 &#181;L of 100 mM ATP, 4.50 &#181;L of 10 mM GTP, 1.13 &#181;L of 200 &#181;M Taxol, and 1.57 &#181;L of 20 &#181;M 488-phalloidin. Ensure the total volume is 25 &#181;L.</li>
	</ol>
	</li>
	<li>Follow steps 4.3.2-4.5 with the specific concentrations for a crosslinker:actin molar ratio of RA = 0.02 and crosslinker:tubulin molar ratio of RT =&#160;0.005. These RA and RT values result in similar lengths between crosslinkers along actin filaments and microtubules (dA <img alt="Equation 1" src="/files/ftp_upload/64228/64228eq1.png" />&#160;60 nm and dMT&#160;<img alt="Equation 1" src="/files/ftp_upload/64228/64228eq1.png" /> 67 nm), estimated using dA = Imonomer/2RA, where Imonomer is the length of an actin monomer, and dMT = Iring/26RT, where Iring is the length of a ring of 13 tubulins15,17.</li>
</ol>

<ol>
	<li>Fletcher, D. A., Mullins, R. D. Cell mechanics and the cytoskeleton. Nature. 463 (7280), 485-492 (2010).</li>
	<li>Koenderink, G. H., Paluch, E. K. Architecture shapes contractility in actomyosin networks. Current Opinion in Cell Biology. 50, 79-85 (2018).</li>
	<li>Dogterom, M., Koenderink, G. H. Actin-microtubule crosstalk in cell biology. Nature Reviews Molecular Cell Biology. 20 (1), 38-54 (2019).</li>
	<li>Burla, F., Mulla, Y., Vos, B. E., Aufderhorst-Roberts, A., Koenderink, G. H. From mechanical resilience to active material properties in biopolymer networks. Nature Reviews Physics. 1 (4), 249-263 (2019).</li>
	<li>Wen, Q., Janmey, P. A. Polymer physics of the cytoskeleton. Current Opinion in Solid State and Materials Science. 15 (5), 177-182 (2011).</li>
	<li>Xiao, Q., Hu, X., Wei, Z., Tam, K. Y. Cytoskeleton molecular motors: structures and their functions in neuron. International Journal of Biological Sciences. 12 (9), 1083-1092 (2016).</li>
	<li>Ajeti, V. et al. Wound healing coordinates actin architectures to regulate mechanical work. Nature Physics. 15 (7), 696-705 (2019).</li>
	<li>Jung, W. et al. Dynamic motions of molecular motors in the actin cytoskeleton. Cytoskeleton. 76 (11-12), 517-531 (2019).</li>
	<li>Pollard, T. D., O&#39;Shaughnessy, B. Molecular mechanism of cytokinesis. Annual Review of Biochemistry. 88 (1), 661-689 (2019).</li>
	<li>Huber, F., Boire, A., L&#243;pez, M. P., Koenderink, G. H. Cytoskeletal crosstalk: when three different personalities team up. Current Opinion in Cell Biology. 32, 39-47 (2015).</li>
	<li>Rivero, F. et al. The role of the cortical cytoskeleton: F-actin crosslinking proteins protect against osmotic stress, ensure cell size, cell shape and motility, and contribute to phagocytosis and development. Journal of Cell Science. 109 (11), 2679-2691 (1996).</li>
	<li>Duclos, G. et al. Topological structure and dynamics of three-dimensional active nematics. Science. 367 (6482), 1120-1124 (2020).</li>
	<li>Baclayon, M. et al. Optical tweezers-based measurements of forces and dynamics at microtubule ends. Optical Tweezers. 1486, 411-435 (2017).</li>
	<li>Gurmessa, B., Fitzpatrick, R., Falzone, T. T., Robertson-Anderson, R. M. Entanglement density tunes microscale nonlinear response of entangled actin. Macromolecules. 49 (10), 3948-3955 (2016).</li>
	<li>Francis, M. L. et al. Non-monotonic dependence of stiffness on actin crosslinking in cytoskeleton composites. Soft Matter. 15 (44), 9056-9065 (2019).</li>
	<li>Ricketts, S. N. et al. Varying crosslinking motifs drive the mesoscale mechanics of actin-microtubule composites. Scientific Reports. 9 (1), 12831 (2019).</li>
	<li>Lee, G. et al. Active cytoskeletal composites display emergent tunable contractility and restructuring. Soft Matter. 17 (47), 10765-10776 (2021).</li>
	<li>Murrell, M. P., Gardel, M. L. F-actin buckling coordinates contractility and severing in a biomimetic actomyosin cortex. Proceedings of the National Academy of Sciences. 109 (51), 20820-20825 (2012).</li>
	<li>Soares e Silva, M. et al. Active multistage coarsening of actin networks driven by myosin motors. Proceedings of the National Academy of Sciences. 108 (23), 9408-9413 (2011).</li>
	<li>Sonn-Segev, A., Bernheim-Groswasser, A., Roichman, Y. Dynamics in steady state in vitro acto-myosin networks. Journal of Physics: Condensed Matter. 29 (16), 163002 (2017).</li>
	<li>Ideses, Y., Sonn-Segev, A., Roichman, Y., Bernheim-Groswasser, A. Myosin II does it all: assembly, remodeling, and disassembly of actin networks are governed by myosin II activity. Soft Matter. 9 (29), 7127 (2013).</li>
	<li>F&#252;rthauer, S. et al. Self-straining of actively crosslinked microtubule networks. Nature Physics. 15 (12), 1295-1300 (2019).</li>
	<li>Lemma, L. M. et al. Multiscale microtubule dynamics in active nematics. Physical Review Letters. 127 (14), 148001 (2021).</li>
	<li>Fan, Y., Wu, K.-T., Aghvami, S. A., Fraden, S., Breuer, K. S. Effects of confinement on the dynamics and correlation scales in kinesin-microtubule active fluids. Physical Review E. 104 (3), 034601 (2021).</li>
	<li>Triclin, S. et al. Self-repair protects microtubules from destruction by molecular motors. Nature Materials. 20 (6), 883-891 (2021).</li>
	<li>Lee, G. et al. Myosin-driven actin-microtubule networks exhibit self-organized contractile dynamics. Science Advances. 7 (6), eabe4334 (2021).</li>
	<li>Ricketts, S. N., Ross, J. L., Robertson-Anderson, R. M. Co-entangled actin-microtubule composites exhibit tunable stiffness and power-law stress relaxation. Biophysical Journal. 115 (6), 1055-1067 (2018).</li>
	<li>Bendix, P. M. et al. A quantitative analysis of contractility in active cytoskeletal protein networks. Biophysical Journal. 94 (8), 3126-3136 (2008).</li>
	<li>Linsmeier, I. et al. Disordered actomyosin networks are sufficient to produce cooperative and telescopic contractility. Nature Communications. 7 (1), 12615 (2016).</li>
	<li>Stam, S. et al. Filament rigidity and connectivity tune the deformation modes of active biopolymer networks. Proceedings of the National Academy of Sciences. 114 (47), E10037-E10045 (2017).</li>
	<li>Yadav, V. et al. Filament nucleation tunes mechanical memory in active polymer networks. Advanced Functional Materials. 29 (49), 1905243 (2019).</li>
	<li>Ennomani, H. et al. Architecture and connectivity govern actin network contractility. Current Biology. 26 (5), 616-626 (2016).</li>
	<li>Alvarado, J., Sheinman, M., Sharma, A., MacKintosh, F. C., Koenderink, G. H. Molecular motors robustly drive active gels to a critically connected state. Nature Physics. 9 (9), 591-597 (2013).</li>
	<li>Alvarado, J., Cipelletti, L., Koenderink, G. H. Uncovering the dynamic precursors to motor-driven contraction of active gels. Soft Matter. 15 (42), 8552-8565 (2019).</li>
	<li>Jung, W., Murrell, M. P., Kim, T. F-actin cross-linking enhances the stability of force generation in disordered actomyosin networks. Computational Particle Mechanics. 2 (4), 317-327 (2015).</li>
	<li>Lenz, M., Thoresen, T., Gardel, M. L., Dinner, A. R. Contractile units in disordered actomyosin bundles arise from f-actin buckling. Physical Review Letters. 108 (23), 238107 (2012).</li>
	<li>Memarian, F.L. et al. Active nematic order and dynamic lane formation of microtubules driven by membrane-bound diffusing motors. Proceedings of the National Academy of Sciences. 118 (52), e2117107118 (2021).</li>
	<li>Needleman, D., Dogic, Z. Active matter at the interface between materials science and cell biology. Nature Reviews Materials. 2 (9), 17048 (2017).</li>
	<li>Foster, P. J., F&#252;rthauer, S., Shelley, M. J., Needleman, D. J. Active contraction of microtubule networks. eLife. 4, e10837 (2015).</li>
	<li>Thijssen, K. et al. Submersed micropatterned structures control active nematic flow, topology, and concentration. Proceedings of the National Academy of Sciences. 118 (38), e2106038118 (2021).</li>
	<li>Sanchez, T., Chen, D. T. N., DeCamp, S. J., Heymann, M., Dogic, Z. Spontaneous motion in hierarchically assembled active matter. Nature. 491 (7424), 431-434 (2012).</li>
	<li>Colen, J. et al. Machine learning active-nematic hydrodynamics. Proceedings of the National Academy of Sciences. 118 (10), e2016708118 (2021).</li>
	<li>Mitchell, K. A., Tan, A. J., Arteaga, J., Hirst, L. S. Fractal generation in a two-dimensional active-nematic fluid. Chaos: An Interdisciplinary Journal of Nonlinear Science. 31 (7), 073125 (2021).</li>
	<li>Pandolfi, R. J., Edwards, L., Johnston, D., Becich, P., Hirst, L. S. Designing highly tunable semiflexible filament networks. Physical Review E. 89 (6), 062602 (2014).</li>
	<li>Tan, A. J. et al. Topological chaos in active nematics. Nature Physics. 15 (10), 1033-1039 (2019).</li>
	<li>Roostalu, J., Rickman, J., Thomas, C., N&#233;d&#233;lec, F., Surrey, T. Determinants of polar versus nematic organization in networks of dynamic microtubules and mitotic motors. Cell. 175 (3), 796-808.e14 (2018).</li>
	<li>Ndlec, F. J., Surrey, T., Maggs, A. C., Leibler, S. Self-organization of microtubules and motors. Nature. 389 (6648), 305-308 (1997).</li>
	<li>Sheung, J. Y. et al. Motor-driven restructuring of cytoskeleton composites leads to tunable time-varying elasticity. ACS Macro Letters. 10 (9), 1151-1158 (2021).</li>
	<li>McGorty, R. PyDDM v0.2.0. Zenodo. (2022).</li>
	<li>Achiriloaie, D. H. et al. Kinesin and myosin motors compete to drive rich multi-phase dynamics in programmable cytoskeletal composites. arXiv. (2021).</li>
	<li>Wulstein, D. M., Regan, K. E., Garamella, J., McGorty, R. J., Robertson-Anderson, R. M. Topology-dependent anomalous dynamics of ring and linear DNA are sensitive to cytoskeleton crosslinking. Science Advances. 5 (12), eaay5912 (2019).</li>
	<li>McGorty, R. Image-Correlation. at &#60;https://github.com/rmcgorty/Image-Correlation&#62;. (2020).</li>
	<li>Robertson, C. Theory and practical recommendations for autocorrelation-based image correlation spectroscopy. Journal of Biomedical Optics. 17 (8), 080801 (2012).</li>
	<li>McGorty, R. Differential Dynamic Microscopy - Python. at &#60;https://github.com/rmcgorty/Differential-Dynamic-Microscopy---Python&#62;. (2021).</li>
	<li>Cerbino, R., Trappe, V. Differential dynamic microscopy: probing wave vector dependent dynamics with a microscope. Physical Review Letters. 100 (18), 188102 (2008).</li>
	<li>Robertson-Anderson, R. M. Optical tweezers microrheology: from the basics to advanced techniques and applications. ACS Macro Letters. 7 (8), 968-975 (2018).</li>
	<li>Pollard, T. D. Polymerization of ADP-actin. Journal of Cell Biology. 99 (3), 769-777 (1984).</li>
	<li>Cou&#233;, M., Brenner, S. L., Spector, I., Korn, E. D. Inhibition of actin polymerization by latrunculin A. FEBS Letters. 213 (2), 316-318 (1987).</li>
	<li>Pollard, T. D. Actin and actin-binding proteins. Cold Spring Harbor Perspectives in Biology. 8 (8), a018226 (2016).</li>
	<li>Kumar, N. Taxol-induced polymerization of purified tubulin. Mechanism of action. Journal of Biological Chemistry. 256 (20), 10435-10441 (1981).</li>
	<li>K&#228;s, J. et al. F-actin, a model polymer for semiflexible chains in dilute, semidilute, and liquid crystalline solutions. Biophysical Journal. 70 (2), 609-625 (1996).</li>
	<li>Viamontes, J., Narayanan, S., Sandy, A. R., Tang, J. X. Orientational order parameter of the nematic liquid crystalline phase of F -actin. Physical Review E. 73 (6), 061901 (2006).</li>
	<li>Hitt, A. L., Cross, A. R., Williams, R. C. Microtubule solutions display nematic liquid crystalline structure. Journal of Biological Chemistry. 265 (3), 1639-1647 (1990).</li>
	<li>Andexer, J. N., Richter, M. Emerging enzymes for ATP regeneration in biocatalytic processes. ChemBioChem. 16 (3), 380-386 (2015).</li>
	<li>Farhadi, L. et al. Actin and microtubule crosslinkers tune mobility and control co-localization in a composite cytoskeletal network. Soft Matter. 16 (31), 7191-7201 (2020).</li>
	<li>Falzone, T. T., Lenz, M., Kovar, D. R., Gardel, M. L. Assembly kinetics determine the architecture of &#945;-actinin crosslinked F-actin networks. Nature Communications. 3 (1), 861 (2012).</li>
	<li>Thoresen, T., Lenz, M., Gardel, M. L. Reconstitution of contractile actomyosin bundles. Biophysical Journal. 100 (11), 2698-2705 (2011).</li>
	<li>Sanghvi-Shah, R., Weber, G. F. Intermediate filaments at the junction of mechanotransduction, migration, and development. Frontiers in Cell and Developmental Biology. 5, 81 (2017).</li>
	<li>Shen, Y. et al. Effects of vimentin intermediate filaments on the structure and dynamics of in vitro multicomponent interpenetrating cytoskeletal networks. Physical Review Letters. 127 (10), 108101 (2021).</li>
	<li>Laan, L., Roth, S., Dogterom, M. End-on microtubule-dynein interactions and pulling-based positioning of microtubule organizing centers. Cell Cycle. 11 (20), 3750-3757 (2012).</li>
</ol>

The authors have nothing to disclose

A key advance of the reconstituted system described above is its modularity and tunability, so users are encouraged to modify the concentrations of proteins, motors, crosslinkers, etc. to suit their desired outcomes, whether it be to emulate a particular cellular process or engineer a material with specific functionality or mechanical properties. Limitations on the concentration range of actin and tubulin are set at the lower limit by the critical concentration needed to polymerize actin (~0.2 &#956;M)57,58,59 and tubulin (~3 - 4&#160;&#956;M)60, and at the upper limit by the transition to nematic alignment of actin filaments (~90 &#956;M)61,62 or microtubules (~35&#160;&#956;M)63. Actin monomers and tubulin dimers should be polymerized into filaments together, rather than mixed together after polymerization, to ensure that they form homogenously interpenetrating percolated networks that synergistically support each other. The novel dynamics that the composites exhibit rely on this interaction. While it is generally important to follow all steps as outlined in the protocol to successfully reproduce the results shown, some steps are more exacting, while others have room to modify and adjust to fit specific needs and available resources.
For example, one important step in ensuring reproducible results is properly preparing and storing the reagents following the guidelines provided in the Table of Materials. Cytoskeletal proteins (actin, tubulin, myosin, kinesin) are labile and should be aliquoted, flash-frozen with liquid nitrogen, and stored at -80 &#176;C in single-use aliquots. Once removed from -80 &#176;C, aliquots should be kept on ice. Cytoskeletal proteins do not reliably retain function after additional freeze-thaw cycles.
Microtubules are more sensitive to depolymerization and denaturing than actin. Once removed from -80 &#176;C, tubulin should be kept on ice before polymerization, and used within 12 h. Once polymerized, microtubules should be kept at room temperature. It is also critical to stabilize microtubules with taxol to prevent depolymerization. Phalloidin-stabilization of actin filaments is likewise important to suppress the ATP-consuming actin treadmilling that competes with myosin and kinesin activity.
Ultracentrifugation of myosin motors is another critical step, as it removes inactive myosin dead heads. Not removing the enzymatically inactive monomers results in passive crosslinking of the actin network and loss of activity. To prolong ATPase activity of motors, an ATP regeneration system such as creatine phosphate and creatine phosphokinase64 can be incorporated.
Finally, maintaining composite activity requires inhibiting adsorption of filaments and motors to the walls of the sample chamber, which can be achieved by passivation of the microscope coverslips and slides. Motor proteins are particularly prone to adsorption, which results in the composite being pulled to the surface of the sample chamber, moving out of the field-of-view, collapsing to 2D, and no longer undergoing activity. Silanizing the coverslips and slides is an effective way to passivate the surfaces and prevent adsorption (see step 1). An alternative passivation method used effectively in in vitro cytoskeleton experiments is coating the surface with a lipid bilayer, similar to the cell membrane18. This method is advantageous if one wishes to tether proteins to the surface or introduce other specific protein-surface interactions, because the bilayer can be functionalized. For optical tweezers experiments, passivation of the microspheres is also critical, and can be achieved by coating carboxylated microspheres with BSA or PEG via carbodiimide crosslinker chemistry48.
There are a few aspects of the presented protocols that researchers may consider altering to suit their needs. Firstly, researchers may choose to replace non-native biotin-NA crosslinkers with biological crosslinkers, such as alpha-actinin or MAP65 that crosslink actin and microtubules, respectively28,65,66. The use of non-native crosslinkers in the composites described here is motivated by their enhanced reproducibility, stability, and tunability compared to native crosslinkers. Because of the strong biotin-NA bond, crosslinkers can be assumed to be permanent, rather than most native crosslinkers that transiently bind with wide-ranging turnover rates. The dynamics of transient crosslinking complicates parsing the contributions from crosslinkers and motors to the dynamics. Moreover, biotin-NA linkers can be versatilely used to crosslink both actin and microtubules, as well as crosslink actin to microtubules. In this way, an unambiguous comparison between crosslinking motifs can be made, keeping all other variables (e.g., crosslinker size, binding affinity, stoichiometry, etc.) fixed. Finally, the reagents needed to incorporate biotin-NA linkers are widely commercially available, well-characterized, and commonly used in many biophysics labs. However, one of the key strengths of the in vitro platform described here is its modularity, so researchers should be able to seamlessly replace biotin-NA linkers with native linkers should they choose.
Secondly, in the current protocol, actin monomers and tubulin dimers are polymerized into filaments together in a centrifuge tube prior to adding to the sample chamber. Flowing the solution of entangled filamentous proteins into the sample chamber may cause flow alignment, especially of the microtubules, which breaks the desired isotropy and homogeneity of the composites. Indeed, a major advance in prior work on steady-state actin-microtubule composites was the ability to co-polymerize actin and microtubules in situ (in the sample chamber) to ensure formation of isotropic interpenetrating networks of actin and microtubules15,16,27. However, extending this approach to active composites would require adding the motors to the sample prior to actin and tubulin polymerization and having the entire sample incubate together at 37 &#176;C prior to experiments. Tests of this variation to the protocol have resulted in reduced actin polymerization and no discernible motor activity, likely due to competing ATPase activity and the prolonged 37&#160;&#176;C incubation of the motors. Fortunately, there is no discernible flow alignment of composites when following the current protocols, as can be seen in Figure 2, Figure 3, and Figure 6. Nevertheless, researchers are encouraged to design protocols that allow for in situ formation of active composites.
Another point of consideration is the fluorescence labeling scheme, which entails sparsely labeling all of the actin filaments and microtubules in the network. This labeling approach was optimized to directly visualize the structure of the network rather than inferring structure and dynamics via tracer filaments or microspheres. However, the tradeoff is that individual filaments are not brightly labeled and resolvable. One approach that researchers could take to both resolve single filaments as well as visualize network structure is to dope in pre-formed filaments labeled with another fluorophore, so both the surrounding network and individual filaments could be imaged simultaneously. However, when using more than two fluorophores and excitation/emission channels, bleed-through between channels is often hard to eliminate, so care must be taken in choosing the fluorophores, filters, and laser intensities.
A related limitation is the inability to visualize the myosin or kinesin motors in the composites. The fluorescent-labeled actin monomers and tubulin dimers used are commercially available, whereas visualization of myosin or kinesin in composites requires in-house labeling. Researchers are encouraged to take the next step to label motors, as done previously18,67, to be able to unequivocally link motor activity and binding to the dynamics and structures that our composites exhibit.
Finally, it is important to note that, in the current protocol, the onset and duration of kinesin activity is not controlled. Because the myosin activity is controlled using photo-deactivation of blebbistatin, as described above, to build in similar light-activation of kinesin, one can incorporate light-activated ATP.
To build up the complexity of the designs described here, to better mimic cellular conditions and broaden the dynamic-structure-function parameter space, future work will focus on incorporating intermediate filaments, such as vimentin68,69, as well as other motors such as dynein13,70. Gelsolin will also be incorporated at different concentrations to control actin length14, as well as tau protein to control microtubule stiffness.
In summary, the presented protocols describe how to design, create, and characterize the dynamics, structure, and mechanics of cytoskeleton-inspired active matter systems, that contain two separate active force-generating components that act on different substrates in a single system. This tunable and modular platform brings reconstitution efforts one important step closer to mimicking the cellular cytoskeleton and offers the unique ability to program its properties across a wide phase space by independently incorporating, removing, and tuning the different components. Moreover, all components of this versatile system are commercially available (see Table of Materials), except for the kinesin dimers which are purified in the Ross Lab, as described previously50, and available upon request. Finally, all analysis code is freely available through GitHub49 and is based on free programming languages and software (Python and Fiji). The transparent dissemination of protocols to design these systems will hopefully make this platform more accessible to a diverse group of users with different expertise, backgrounds, institutional affiliations, and research goals.

An erratum was issued for: <a href="https://www.jove.com/t/64228">Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and Mechanics</a>. The Authors section was updated.
Mehrzad Sasanpour1 
Daisy H. Achiriloaie1,2 
Gloria Lee1 
Gregor Leech1 
Christopher Currie1 
K. Alice Lindsay3 
Jennifer L. Ross3 
Ryan J. McGorty1 
Rae M. Robertson-Anderson1 
1Department of Physics and Biophysics, University of San Diego 
2W. M. Keck Science Department, Scripps College, Pitzer College, and Claremont McKenna College 
3Department of Physics, Syracuse University
to:
Mehrzad Sasanpour1 
Daisy H. Achiriloaie1,2 
Gloria Lee1 
Gregor Leech1 
Maya Hendija1 
K. Alice Lindsay3 
Jennifer L. Ross3 
Ryan J. McGorty1 
Rae M. Robertson-Anderson1 
1Department of Physics and Biophysics, University of San Diego 
2W. M. Keck Science Department, Scripps College, Pitzer College, and Claremont McKenna College 
3Department of Physics, Syracuse University

An erratum was issued for:&#160;<a href="https://www.jove.com/t/64228">Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and Mechanics</a>. The Authors section was updated.

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

The cytoskeleton is a dynamic composite network of interacting biopolymers that provides structural and mechanical support to cells. Associated molecular motors and binding proteins restructure and adapt the cytoskeleton to allow cells to grow, change shape, stiffen, move, and even self-heal, enabling myriad cellular processes ranging from migration and division to mechanosensing1,2. Beyond its significance in cellular biophysics, the cytoskeleton is also a quintessential example of active matter with potential materials applications ranging from wound healing and drug delivery to filtration and soft robotics1,3,4,5,6,7,8,9.
The two key characteristics that endow the cytoskeleton with its unique structural and mechanical diversity and multifunctionality are: 1) its composite nature, comprising multiple interacting protein filaments, such as semiflexible actin filaments and rigid microtubules, as well as their associated binding and crosslinking proteins3,5,10; and 2) its ability to continuously restructure, move, coarsen, and perform work via energy-consuming motors, such as myosins and kinesins, pushing and pulling on the filamentous proteins1,7,11,12,13. While this elegant complexity enables the cytoskeleton to mediate processes as diverse as cell motility, cytokinesis, and wound-healing3,6,7,11, it hampers the ability of researchers to reproduce the signature in vivo characteristics of the cytoskeleton in reconstituted in vitro systems.
Current frontier reconstitution efforts focus on composites of entangled and crosslinked actin filaments and microtubules3,10,14,15,16,17, force-generating actomyosin networks2,8,18,19,20,21, and active nematics driven by kinesin-microtubule interactions22,23,24,25,26. Steady-state actin-microtubule composites have been shown to display emergent mechanical properties15,16,27, such as enhanced filament mobility and increased stiffness compared to single-component systems27. Studies on in vitro actomyosin systems have reported a wide range of structural and dynamical properties that depend on the concentrations of actin, myosin, and crosslinkers28,29,30,31. For example, with sufficient crosslinking, actomyosin networks undergo large-scale contraction and coarsening2,28,30,32,33,34,35,36, whereas without crosslinkers, networks display rapid, destabilizing flow and rupturing19,29. Reconstituted microtubule-based active nematics that use clusters of kinesin motors to crosslink and pull on microtubule bundles have been reported to exhibit long lasting turbulent flows, extension, buckling, fracturing, and healing12,22,23,24,25,37,38,39,40,41,42,43,44,45,46,47.
More recently, actin-microtubule composites driven by myosin II mini-filaments have been shown to lead to more ordered contraction and network integrity compared to the disordered flow and network rupturing that actomyosin networks without crosslinkers exhibit17,26,48. Moreover, the combination of composite robustness and force-generation is optimized when actin and microtubules are present at comparable concentrations. Key emergent features in this region of formulation space include enhanced mechanical strength26, coordinated motion of actin and microtubules26, steady sustained contraction, and mesoscale restructuring17.
Here, protocols are described to engineer and tune co-entangled and crosslinked composites of microtubules and actin filaments that are pushed out of equilibrium by myosin II mini-filaments and kinesin clusters acting on actin filaments and microtubules, respectively (Figure 1). The dynamics, structure, and mechanics of this class of composites can be tuned by the relative concentrations of the filaments, motors, and crosslinkers to exhibit a rich phase space of advective and turbulent flow, isotropic contraction, acceleration, deceleration, de-mixing, stiffening, relaxation, and rupturing. The focus of this work is on preparing and tuning this class of active cytoskeletal composites. However, to aid researchers in benchmarking and characterizing the described active composites, effective imaging methods using multi-spectral confocal microscopy are also detailed. Finally, results of key computational analysis methods that can be used to measure the dynamics, structure, and mechanics of the composites are presented. Researchers are encouraged to adopt these methods-which include differential dynamic microscopy (DDM), spatial image autocorrelation (SIA), and particle image velocimetry (PIV)-as they have been optimized to characterize the complex dynamics and structural diversity of the composites17,26,49.
The steps described below focus on preparing the composites and imaging them using confocal microscopy. Protocols describing post-acquisition data analysis and optical tweezers measurements can be found in previous works&#160;17,26,48,50,&#160;and provided upon request. All materials are listed in the Table of Materials provided.

<table><tbody><tr><td>(-)-Blebbistatin&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; blebbistatin</td><td>Sigma Aldrich</td><td>B0560</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 200 &amp;mu;M in DMSO&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; dessicated, in DMSO, -20&amp;ordm;C&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; dissolve 1 mg of powder to 200 &amp;mu;M in DMSO&lt;br/&gt; &lt;strong&gt;Storage, Handling, Troubleshooting Notes:&lt;/strong&gt; limited shelf-life, typically stops functioning reliably after 3-4 months. purchase and prepare new solution every 3 months.&amp;nbsp;</td></tr><tr><td>1:20 488-tubulin:tubulin mixture&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; 5-488-tubulin</td><td>NA</td><td>NA</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 5 mg/ml in PEM&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; single use aliquots, -80&amp;ordm;C, avoid freeze-thaw cycles&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; mix tubulin and 488-tubulin at a 20:1 ratio, flash freeze with LN2&lt;br/&gt; &lt;strong&gt;Storage, Handling, Troubleshooting Notes:&lt;/strong&gt; each aliquot can be used for up to 12 hrs stored on ice at 4&amp;ordm;C, protect from light</td></tr><tr><td>1:20 R-tubulin:tubulin mixture&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; 5-R-tubulin</td><td>NA</td><td>NA</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 5 mg/ml in PEM&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; single use aliquots, -80&amp;ordm;C, avoid freeze-thaw cycles&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; mix tubulin and rhodamine tubulin at a 20:1 ratio, flash freeze with LN2&lt;br/&gt; &lt;strong&gt;Storage, Handling, Troubleshooting Notes:&lt;/strong&gt; each aliquot can be used for up to 12 hrs stored on ice at 4&amp;ordm;C, protect from light</td></tr><tr><td>actin (biotin): skeletal muscle&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; biotin-actin</td><td>Cytoskeleton</td><td>AB07</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 1 mg/ml in G-buffer&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; single use aliquots, -80&amp;ordm;C, avoid freeze-thaw cycles&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; reconstitute to 1 mg/ml in G-buffer, flash freeze with LN2&lt;br/&gt; &lt;strong&gt;Storage, Handling, Troubleshooting Notes:&lt;/strong&gt; (1) immediately prior to use dilute to 0.5 mg/ml in PEM, (2) once removed from -80&amp;ordm;C, store&amp;nbsp; aliquot on ice at 4&amp;ordm;C for up to 1 week</td></tr><tr><td>actin (rhodamine): rabbit skeletal muscle&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; R-actin</td><td>Cytoskeleton</td><td>AR05</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 1.5 mg/ml in G-buffer&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; single use aliquots, -80&amp;ordm;C, avoid freeze-thaw cycles&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; reconstitute to 1.5 mg/ml in G-buffer, flash freeze with LN2&lt;br/&gt; &lt;strong&gt;Storage, Handling, Troubleshooting Notes:&lt;/strong&gt; once removed from -80&amp;ordm;C, store&amp;nbsp; aliquot on ice at 4&amp;ordm;C, can be used for up to 1 week</td></tr><tr><td>adenosine triphosphate&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; ATP</td><td>Thermo Fisher Scientific</td><td>A1048</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 100 mM&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; in solution (pH 7), -20&amp;ordm;C&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; reconsitute in DI H&lt;sub&gt;2&lt;/sub&gt;0, bring pH to 7 with NaOH&lt;br/&gt; &lt;strong&gt;Storage, Handling, Troubleshooting Notes:&lt;/strong&gt; routinely check pH and adjust as needed, hydrolyzes over time, replace every ~6-12 months</td></tr><tr><td>AlexaFluor488 Phalloidin&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; 488-phalloidin</td><td>Thermo Fisher Scientific</td><td>A12379</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 100 &amp;mu;M DMSO&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; protected from light, dessicated, -20&amp;ordm;C&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; reconstitute to 100 &amp;mu;M with DMSO&lt;br/&gt; &lt;strong&gt;Storage, Handling, Troubleshooting Notes:&lt;/strong&gt; immediately prior to use dilute to 20 &amp;mu;M in PEM (1 &amp;mu;L in 4 &amp;mu;L PEM)</td></tr><tr><td>AlexaFluor488&amp;ndash;labeled actin&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; 488-actin</td><td>Thermo Fisher Scientific</td><td>A12373</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 1.5 mg/ml in G-buffer&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; single use aliquots, -80&amp;ordm;C, avoid freeze-thaw cycles&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; reconstitute to 1.5 mg/ml in G-buffer, flash freeze with LN2&lt;br/&gt; &lt;strong&gt;Storage, Handling, Troubleshooting Notes:&lt;/strong&gt; this item has been discontinued</td></tr><tr><td>Basic Plasma Cleaner&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; plasma cleaner</td><td>Harrick Plasma</td><td>PDC-32G</td><td></td></tr><tr><td>Bemis&amp;nbsp;Parafilm M Laboratory Wrapping Film&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; transparent film</td><td>Thermo Fisher Scientific</td><td>13-374-5</td><td></td></tr><tr><td>D-(+)-Glucose&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt;&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A1682836</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 100x&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; store at stock concentration (100x) or 10x concentration, dessicated, at -20&amp;ordm;C&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; reconstitute powder to 4.5 mg/ml in DI H&lt;sub&gt;2&lt;/sub&gt;0&lt;br/&gt; &lt;strong&gt;Storage, Handling, Troubleshooting Notes:&lt;/strong&gt; final concentration in solution should 45 &amp;mu;g/mL&amp;nbsp;</td></tr><tr><td>D-Biotin&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; biotin</td><td>Fisher Scientific</td><td>BP232-1</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 1.02 mM in PEM&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; dessicated, 4&amp;ordm;C</td></tr><tr><td>deionized nanopure water&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; DI</td><td></td><td></td><td></td></tr><tr><td>Dimethyldichlorosilane&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; silane</td><td>Thermo Fisher Scientific</td><td>D/3820/PB05</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 2% dissolved in Toulene</td></tr><tr><td>Dithiothreitol&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; DTT</td><td>Thermo Fisher Scientific</td><td>R0861</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 1 M in DMSO&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; dessicated, -20&amp;ordm;C&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; dilute to 2 mM in PEM immediately before each experiment</td></tr><tr><td>DMSO Anhydrous&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; DMSO</td><td>Thermo Fisher Scientific</td><td>D12345</td><td></td></tr><tr><td>F-Buffer&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; F-buffer</td><td>NA</td><td>NA</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 10x&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; dessicated, -20&amp;ordm;C&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; 10 mM Imidazole (pH 7.0), 50 mM KCl, 1 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 1 mM EGTA, 0.2 mM ATP</td></tr><tr><td>G-Buffer&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; G-buffer</td><td>NA</td><td>NA</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 10x&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; dessicated, -20&amp;ordm;C&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; 2.0 mM Tris (pH 8), 0.2 mM ATP, 0.5 mM DTT, 0.1 mM CaCl&lt;sub&gt;2&lt;/sub&gt;. Store at -20&amp;deg;C.</td></tr><tr><td>glass microscope slide&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; slide</td><td>Thermo Fisher Scientific</td><td>22-310397</td><td></td></tr><tr><td>Glucose oxidase + catalase + &amp;beta;-mercaptoethanol&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; GOC</td><td>Sigma Aldrich</td><td>G2133-250KU, C1345, 63689&amp;nbsp;</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 100x&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; store at stock concentration (100x) or 10x concentration, dessicated, at -20&amp;ordm;C&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; For 100x: 4.3 mg/ml glucose oxidase, 0.7 mg/ml catalase, 0.5% v/v&amp;nbsp; &amp;beta;-mercaptoethanol in DI H&lt;sub&gt;2&lt;/sub&gt;0&lt;br/&gt; &lt;strong&gt;Storage, Handling, Troubleshooting Notes:&lt;/strong&gt; final concentration in solution should be: 0.005% &amp;beta;-mercaptoethanol, 43 &amp;mu;g/mL glucose oxidase, 7 &amp;mu;g/mL catalase</td></tr><tr><td>glu-GOC oxygen scavenging system&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; glu-GOC</td><td>NA</td><td>NA</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 100x&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; prepare fresh each time&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; mix equal parts Glu and GOC and add at 1/100 final sample volume immediately before imaging&lt;br/&gt; &lt;strong&gt;Storage, Handling, Troubleshooting Notes:&lt;/strong&gt; prepare from Glu and GOC immediately before imaging</td></tr><tr><td>Guanosine triphosphate&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; GTP</td><td>Thermo Fisher Scientific</td><td>R0461</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 100 mM&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; 100 &amp;mu;L aliquots at -20&amp;ordm;C</td></tr><tr><td>Instant Mix 1-minute epoxy&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; epoxy</td><td>Loctite</td><td>1366072&amp;nbsp;</td><td></td></tr><tr><td>Kinesin-1 401 BIO 6x HIS&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; kinesin</td><td>Prepared in JL Ross Lab at Syracuse University</td><td>NA</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 8.87 &amp;mu;M in PEM&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; single use aliquots, -80&amp;ordm;C, avoid freeze-thaw cycles&lt;br/&gt; &lt;strong&gt;Storage, Handling, Troubleshooting Notes:&lt;/strong&gt; biotinylated dimers form kinesin clusters, each aliquot can be used for up to 12 hrs stored on ice at 4&amp;ordm;C</td></tr><tr><td>NeutrAvidin&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; NA</td><td>Thermo Fisher Scientific</td><td>31000</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 5 mg/ml in PEM&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; dessicated, -20&amp;ordm;C&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; reconstitute powder to 5 mg/ml in PEM</td></tr><tr><td>No 1. glass coverslips (24 mm x 24 mm)&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; coverslip</td><td>Thermo Fisher Scientific</td><td>12-548-CP</td><td></td></tr><tr><td>Paclitaxel&amp;nbsp;&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; Taxol</td><td>Thermo Fisher Scientific</td><td>P3456</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 2 mM in DMSO&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; protected from light, dessicated, -20&amp;ordm;C&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; reconstitute to 2 mM with DMSO&lt;br/&gt; &lt;strong&gt;Storage, Handling, Troubleshooting Notes:&lt;/strong&gt; immediately prior to use dilute to 200 &amp;mu;M in DMSO (0.4 &amp;mu;L in 3.6 &amp;mu;L DMSO)</td></tr><tr><td>PEM-100&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; PEM</td><td>NA</td><td>NA</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 1x&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; room temperature (RT)&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; 100 mM K-PIPES (pH 6.8), 2 mM EGTA, 2 mM MgCl&lt;sub&gt;2&lt;/sub&gt;&lt;br/&gt; &lt;strong&gt;Storage, Handling, Troubleshooting Notes:&lt;/strong&gt; use KOH to adjust pH to 6.8, recheck pH often and adjust accordingly</td></tr><tr><td>phalloidin&lt;br/&gt; &lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; phalloidin</td><td>Thermo Fisher Scientific</td><td>P3457</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 100 &amp;mu;M in DMSO&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; protected from light, dessicated, -20&amp;ordm;C, adhere closely to storage/handling conditions&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; reconstitute to 100 &amp;mu;M with DMSO&lt;br/&gt; &lt;strong&gt;Storage, Handling, Troubleshooting Notes:&lt;/strong&gt; susceptible to impurities in its preparation and denaturing, identifiable as large amorphous aggregates of actin in samples</td></tr><tr><td>porcine brain tubulin&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; tubulin</td><td>Cytoskeleton</td><td>T240</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 5 mg/ml in PEM&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; single use aliquots, -80&amp;ordm;C, avoid freeze-thaw cycles&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; reconstitute powder to 5 mg/ml in PEM, flash freeze with LN2&lt;br/&gt; &lt;strong&gt;Storage, Handling, Troubleshooting Notes:&lt;/strong&gt; each aliquot can be used for up to 12 hrs stored on ice at 4&amp;ordm;C</td></tr><tr><td>Potassium Chloride&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; KCl</td><td>Thermo Fisher Scientific</td><td>AM9640G</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 4 M&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; RT</td></tr><tr><td>Rabbit skeletal actin&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; actin</td><td>Cytoskeleton</td><td>AKL99</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 2 mg/ml in G-buffer&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; single use aliquots, -80&amp;ordm;C, avoid freeze-thaw cycles&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; reconstitute to 2 mg/ml in G-buffer, flash freeze with LN2&lt;br/&gt; &lt;strong&gt;Storage, Handling, Troubleshooting Notes:&lt;/strong&gt; once removed from -80&amp;ordm;C, store&amp;nbsp; aliquot on ice at 4&amp;ordm;C, can be used for up to 1 week</td></tr><tr><td>Rabbit skeletal myosin II&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; myosin</td><td>Cytoskeleton</td><td>MY02</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 10 mg/ml in PEM&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; single use aliquots, -80&amp;ordm;C, avoid freeze-thaw cycles&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; reconstitute powder to 10 mg/ml in PEM, flash freeze with LN2&lt;br/&gt; &lt;strong&gt;Storage, Handling, Troubleshooting Notes:&lt;/strong&gt; monomers form minifilaments at low KCl, each aliquot can be used for up to 12 hrs stored on ice at 4&amp;ordm;C</td></tr><tr><td>Tubulin (biotin): porcine brain&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; biotin-tubulin</td><td>Cytoskeleton</td><td>T333P</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 5 mg/ml in PEM&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; single use aliquots, -80&amp;ordm;C, avoid freeze-thaw cycles&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; reconstitute powder to 5 mg/ml in PEM, flash freeze with LN2&lt;br/&gt; &lt;strong&gt;Storage, Handling, Troubleshooting Notes:&lt;/strong&gt; immediately prior to use dilute to 0.5 mg/ml in PEM</td></tr><tr><td>Tubulin (fluorescent HiLyte 488): porcine brain&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; 488-tubulin</td><td>Cytoskeleton</td><td>TL488M</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 5 mg/ml in PEM&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; single use aliquots, -80&amp;ordm;C, avoid freeze-thaw cycles&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; reconstitute powder to 5 mg/ml in PEM, flash freeze with LN2&lt;br/&gt; &lt;strong&gt;Storage, Handling, Troubleshooting Notes:&lt;/strong&gt; each aliquot can be used for up to 12 hrs stored on ice at 4&amp;ordm;C, protect from light</td></tr><tr><td>tubulin (rhodamine): porcine brain&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; R-tubulin</td><td>Cytoskeleton</td><td>TL590M</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 5 mg/ml in PEM&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; single use aliquots, -80&amp;ordm;C, avoid freeze-thaw cycles&lt;br/&gt; &lt;strong&gt;Stock and Experiment Recipes:&lt;/strong&gt; reconstitute powder to 5 mg/ml in PEM, flash freeze with LN2&lt;br/&gt; &lt;strong&gt;Storage, Handling, Troubleshooting Notes:&lt;/strong&gt; each aliquot can be used for up to 12 hrs stored on ice at 4&amp;ordm;C, protect from light</td></tr><tr><td>Tween 20&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; Tween20</td><td>Thermo Fisher Scientific</td><td>J20605.AP</td><td>&lt;strong&gt;Stock Concentration:&lt;/strong&gt; 1% v/v in DI H&lt;sub&gt;2&lt;/sub&gt;0&lt;br/&gt; &lt;strong&gt;Storage:&lt;/strong&gt; RT</td></tr><tr><td>ultracentrifuge grade microtubes&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; Beckman-Coulter Optima Max XP&amp;nbsp;</td><td>Beckman Coultier</td><td>343776</td><td>&lt;strong&gt;Storage, Handling, Troubleshooting Notes:&lt;/strong&gt; 8x34 mm PC</td></tr><tr><td>UV light curing glue&lt;br/&gt; &amp;nbsp;&lt;strong&gt;Abbreviation used in paper:&lt;/strong&gt; UV glue</td><td>Pharda</td><td>SKG-2869</td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Reconstituting and Characterizing Actin-Microtubule Composites with Tunable Motor-Driven Dynamics and Mechanics at JoVE.com

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

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 ...

reconstituting-characterizing-actin-microtubule-composites-with

Research

JoVE Journal

Biology

3.0K Views.  University of San Diego. 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. 

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

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.

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 ...

Engineering

Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor individual reactions live in well-controlled conditions. To do so, live single-filament experiments have emerged over the past 20 years, mostly using total internal reflection fluorescence (TIRF) microscopy, and have provided a trove of key results. In 2011, in order to further expand the possibilities of these experiments and to avoid recurring problematic artifacts, we introduced simple microfluidics in these assays. This study details our basic protocol, where individual actin filaments are anchored by one end to the passivated coverslip surface, align with the flow, and can be successively exposed to different protein solutions. We also present the protocols for specific applications and explain how controlled mechanical forces can be applied, thanks to the viscous drag of the flowing solution. We highlight the technical caveats of these experiments and briefly present possible developments based on this technique. These protocols and explanations, along with today&#39;s availability of easy-to-use microfluidics equipment, should allow non-specialists to implement this assay in their labs.

We present protocols for simple actin filament microfluidic assays, in combination with fluorescence microscopy, that allow one to accurately monitor individual actin filaments in real-time while sequentially exposing them to different protein solutions.

In order to decipher the complex molecular mechanisms that regulate the assembly and disassembly of actin filaments, it is a great asset to monitor ...

Biochemistry

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 ...

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence TIRF Microscopy

This protocol is a guide for visualizing dynamic actin and microtubules using an in vitro total internal fluorescence (TIRF) microscopy assay.

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and ...

Probing Myosin Ensemble Mechanics in Actin Filament Bundles Using Optical Tweezers

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle contraction. To understand their force-generating mechanisms, myosin II has been investigated both at the single-molecule (SM) level and as teams of motors in vitro using biophysical methods such as optical trapping.
These studies showed that myosin force-generating behavior can differ greatly when moving from the single-molecule level in a three-bead arrangement to groups of motors working together on a rigid bead or coverslip surface in a gliding arrangement. However, these assay constructions do not permit evaluating the group dynamics of myosin within viscoelastic structural hierarchy as they would within a cell. We have developed a method using optical tweezers to investigate the mechanics of force generation by myosin ensembles interacting with multiple actin filaments.
These actomyosin bundles facilitate investigation in a hierarchical and compliant environment that captures motor communication and ensemble force output. The customizable nature of the assay allows for altering experimental conditions to understand how modifications to the myosin ensemble, actin filament bundle, or the surrounding environment result in differing force outputs.

Formation of actomyosin bundles in vitro and measuring myosin ensemble force generation using optical tweezers is presented and discussed.

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle ...

Bioengineering

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell deformation, division, migration, and adhesion. However, studying the dynamics and structure of the actin network in vivo is complicated by the biochemical and genetic regulation within live cells. To build a minimal model devoid of intracellular biochemical regulation, actin is encapsulated inside giant unilamellar vesicles (GUVs, also called liposomes). The biomimetic liposomes are cell-sized and facilitate a quantitative insight into the mechanical and dynamical properties of the cytoskeleton network, opening a viable route for bottom-up synthetic biology. To generate liposomes for encapsulation, the inverted emulsion method (also referred to as the emulsion transfer method) is utilized, which is one of the most successful techniques for encapsulating complex solutions into liposomes to prepare various cell-mimicking systems. With this method, a mixture of proteins of interest is added to the inner buffer, which is later emulsified in a phospholipid-containing mineral oil solution to form monolayer lipid droplets. The desired liposomes are generated from monolayer lipid droplets crossing a lipid/oil-water interface. This method enables the encapsulation of concentrated actin polymers into the liposomes with desired lipid components, paving the way for in vitro reconstitution of a biomimicking cytoskeleton network.

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

In this manuscript, we demonstrate the experimental techniques to encapsulate the F-actin cytoskeleton into giant unilamellar lipid vesicles (also called liposomes), and the method to form a cortex-biomimicking F-actin layer at the inner leaflet of the liposome membrane.

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell ...

Reconstitution of Actin-Based Motility with Commercially Available Proteins

Many cell movements and shape changes and certain types of intracellular bacterial and organelle motility are driven by the biopolymer actin that forms a dynamic network at the surface of the cell, organelle, or bacterium. The biochemical and mechanical basis of force production during this process can be studied by reproducing actin-based movement in an acellular manner on inert surfaces such as beads that are functionalized and incubated with a controlled set of components. Under the appropriate conditions, an elastic actin network assembles at the bead surface and breaks open due to the stress generated by network growth, forming an "actin comet" that propels the bead forward. However, such experiments require the purification of a host of different actin-binding proteins, often putting them beyond the reach of non-specialists. This article details a protocol for reproducibly obtaining actin comets and motility of beads using commercially available reagents. Bead coating, bead size, and motility mixture can be altered to observe the effect on bead speed, trajectories, and other parameters. This assay can be used for testing the biochemical activities of different actin-binding proteins, and for performing quantitative physical measurements that shed light on active matter properties of actin networks. This will be a useful tool for the community, enabling the study of in vitro actin-based motility without expert knowledge in actin-binding protein purification.

This protocol describes how to produce actin comets on the surfaces of beads using commercially available protein ingredients. Such systems mimic the protrusive structures found in cells, and can be used to examine physiological mechanisms of force production in a simplified way.

Many cell movements and shape changes and certain types of intracellular bacterial and organelle motility are driven by the biopolymer actin that forms a ...

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 ...

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 ...

Reconstitution of Membrane-Tethered Minimal Actin Cortices on Supported Lipid Bilayers

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane with the underlying actin cortex. In the past decades, reconstituted, minimal systems based on supported lipid bilayers in combination with actin filament networks have proven to be very instrumental in unraveling basic mechanisms and consequences of membrane-tethered actin networks, as well as in studying the functions of individual membrane-associated proteins. Here, we describe how to reconstitute such active composite systems in vitro that consist of fluid supported lipid bilayers coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors that can be readily observed via total internal reflection fluorescence microscopy. An open-chamber design allows one to assemble the system in a step-by-step manner and to systematically control many parameters such as linker protein concentration, actin concentration, actin filament length, actin/myosin ratio, as well as ATP levels. Finally, we discuss how to control the quality of the system, how to detect and troubleshoot commonly occurring problems, and some limitations of this system in comparison with the living cell surface.

This protocol describes the formation of supported lipid bilayers and the addition of cytoskeletal filaments and motor proteins to study the dynamics of reconstituted, membrane-tethered cytoskeletal networks using fluorescence microscopy.

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane ...

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

Summary

Explore More Videos

Chapters in this video

Assembling Molecular Shuttles Powered by Reversibly Attached Kinesins

Using Microfluidics and Fluorescence Microscopy to Study the Assembly Dynamics of Single Actin Filaments and Bundles

Self-Assembly of Microtubule Tactoids

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy

Probing Myosin Ensemble Mechanics in Actin Filament Bundles Using Optical Tweezers

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

Reconstitution of Actin-Based Motility with Commercially Available Proteins

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

Directly Measuring Forces Within Reconstituted Active Microtubule Bundles

Reconstitution of Membrane-Tethered Minimal Actin Cortices on Supported Lipid Bilayers