Our work brings cytoskeleton reconstitution efforts an important step closer to mimicking cellular conditions by engineering composites of actin and microtubules, driven by kinesin and myosin motors to actively tune, restructure, and move. The dynamics and structure of our composites can be precisely programmed by independently adding, removing, and tuning the different components to exhibit a rich phase base of contraction, infection, demising, coarsening, and rupture. Our approach is broadly applicable to design, create, and characterize active matter platforms which incorporate multiple force generating components that act on different substrates in a single system.
Demonstrating the procedure will be Daisy Achiriloaie and Maya Hendija. Undergraduate researchers from our laboratory. To begin, add the reagents to a sterile black 1.5 milliliter micro centrifuge tube using a micro pipette and sterile pipette tips to form Kinesin motor clusters that bind and exert forces between pairs of microtubules.
Mix gently by pipetting the solution up and down. Then incubate the solution for 30 minutes at four degrees Celsius, protect it from light. To prepare a co entangled composite network of acton filaments and microtubules, set the heat block to 37 degrees Celsius.
Add the reagents to a sterile 0.6 milliliter micro centrifuge tube. Ensure that the total volume is 25 microliters. Gently pipette the solution up and down to mix it and place it on the 37 degrees Celsius heat block protected from light for one hour.
After that, remove the tube from the heat block and use a micro pipette to gently mix in 0.84 microliters of 100 micromolar Phalloidin. Incubate for five to 10 minutes at room temperature protected from light. To prepare active composites for conf focal imaging, add the reagents to the solution and mix gently by pipetting up and down.
Divide the solution into three 10 microliter aliquots and label them as K, K plus M, and negative control. Add 2.54 microliters of myosin to the K plus M aliquot and 2.54 microliters of PEM to K and negative control aliquots. Then add 2.5 microliters of Kinesin clusters to K and K plus M aliquots and pipette up and down to mix.
Next, add 2.5 microliters of PEM to the negative control using the same technique. Use a micropipette to slowly flow each solution into the corresponding channel of the sample chambers via capillary action. Push down very slowly and gently on the pipette so as not to introduce air bubbles into the channel.
Seal the two open ends of each channel with fast drying epoxy or UV curable glue. When the adhesive is completely dry, place the channel on the microscope to image the composite as close to the initial inactive state as possible. Image the K channel and K plus M channels first followed by the control channel.
Note the time elapsed between the addition of the Kinesin clusters to K and K plus M aliquots and the beginning of data acquisition. To prepare actin to actin or AA crosslinkers, add Biotin-Actin, NeutrAvidin, Biotin, and PEM to a micro centrifuge tube and mix them gently by pipetting up and down. Similarly, for microtubules to microtubules or MM crosslinkers, add Biotin-tubulin, NeutrAvidin, Biotin, and PEM to a micro centrifuge tube and mix them gently by pipetting up and down.
Wrap the tube in a thermoplastic ceiling film to create a watertight seal and place them in a flotation raft in a temperature controlled sonicater bath, set to four degrees Celsius for 90 minutes. To incorporate A-A crosslinker complexes into samples for imaging, add the reagents to a micro centrifuge tube. Ensure the total volume is 25 microliters.
Similarly, for M-M crosslinker complexes, add the reagents shown on the screen to a micro centrifuge tube. Mix the solution by pipetting up and down and place it on the 37 degree Celsius heat block protected from light for one hour. After that, remove the tube from the heat block and use a micro pipette to mix in 0.84 microliters of 100 micromolar Phalloidin.
Incubate for five to 10 minutes at room temperature protected from light and repeat the procedure shown earlier to prepare active composites for confocal imaging. Actin monofibers and tubulin dimers are co polymerized to form co entangled networks of actin filaments and microtubules. Myosin two mini filaments and Kinesin clusters push and pull on the filaments to drive the composites out of steady state.
Passive cross-linking is achieved using NeutrAvidin to link a biotinylated actin filaments or microtubules. Myosin two mini filaments, Kinesin clusters, or both motors are incorporated into composites with no passive crosslinks, actin-actin crosslinks and microtubule-microtubule crosslinks. Two color confocal imaging of myosin driven cytoskeleton composites with varying myosin concentrations and molar actin fractions is shown here.
Particle image velocimetry shows that actin myosin activity triggers coordinated contractile dynamics of actin and microtubules in co entangled composites. Here, different arrow colors correspond to different speeds as indicated in the color scale to the right of the vector fields. Time resolved differential dynamic microscopy is performed on microtubule and actin channels of time series to determine characteristics of decay times versus wave number for both actin and microtubules.
Contraction speeds are determined via fits to decay time curves, averaged over all lag times for the entire duration of each 45 minute time series. Time resolved differential dynamic microscopy quantifies how the dynamics vary over time by evaluating decay times for consecutive six minute intervals during the 45 minute activation time for actin and microtubules. Time resolved contraction speeds for actin filaments and microtubules are determined from fits to corresponding decay time curves.
Spatial image auto correlation analysis quantifies the motor driven restructuring of active cytoskeletal components by comparing auto correlation curves for the different networks at the beginning of the experiment compared to the end. Time resolved correlation lengths determined via exponential fits of auto correlation curves show composites that do not restructure compared to those that substantially restructure. Two color confocal images of Kinesin driven actin microtubule composites show formulation dependent restructuring over time without crosslinkers composites restructure into loosely connected microtubule rich clusters.
Actin-actin cross-linking supports actin microtubule co-localization, while microtubule-microtubule cross-linking enhances D mixing. Differential dynamic microscopy and spatial image auto correlation analysis show the effect of cross-linking and competing mycin and Kinesin motors on the time varying dynamics and structure of the composites. To mimic cellular conditions more closely, researchers can incorporate intermediate filaments, other motors, and binding proteins that control filament lengths and stiffnesses.
Optical tweezers metrology measurements can also be performed to characterize composite mechanics. Using our approach, researchers can precisely tune the dynamics and structure of cytoskeleton inspired composite active matter across an unprecedented phase space to emulate diverse cellular processes and engineer reconfigurable programmable materials.
