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 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.
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 17,26,48,50, and provided upon request. All materials are listed in the Table of Materials provided.
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.
2. Preparing active actin-microtubule composite driven by myosin mini-filaments
3. Imaging and characterization of active composites using confocal microscopy
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.
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.
To determine successful preparation of active composites (Figure 1), and to characterize their dynamics and structure, a laser scanning fluorescence microscope with at least two fluorescence channels is used to visualize the actin filaments and microtubules simultaneously (Figure 2 and Figure 6). All actin filaments and microtubules in the composites are sparsely labeled, rather than doping in tracer bright filaments, as is often done in in vitro studies. This method ensures that the measured dynamics and structure are representative of the composite itself rather than the tracers which are formed under different conditions than the composites. For this reason, individual actin filaments and microtubules cannot typically be resolved, rather images portray mesoscale network structure (Figure 2 and Figure 6).
This labeling approach was optimized for spatial image autocorrelation (SIA) and differential dynamic microscopy (DDM) analyses that examine the dynamics and structure in reciprocal Fourier space (Figure 4, Figure 5, and Figure 8)52,53,54,55. Particle-image velocimetry (PIV) can also be used to depict and characterize dynamics and flow fields (Figure 3 and Figure 7), but it requires pixel-binning (lower spatial resolution) and larger lag-time increments (lower temporal resolution) than SIA and DDM to eliminate erroneous vectors that arise from noise in the dense, low-signal images. Nevertheless, PIV is recommended for qualitative examination of flow fields and corroboration of DDM results (Figure 4 and Figure 8)26,50.
Sample characterization of the described networks using these analyses (i.e., DDM, SIA, PIV) is provided to aid researchers in adopting similar analyses to benchmark and characterize their samples. However, detailed descriptions of these techniques are outside the scope of this work. For detailed descriptions of how to perform DDM on these and other similar systems, including user-friendly Python code, refer to previous works17,26,49,50 and the references there within. For details regarding how to perform SIA and PIV on the systems described here, the reader is directed to previous works17,50.
Several controls, described below, should be done to ensure that the composites are functioning as expected. A composite without myosin or kinesin should appear essentially static with minimal thermal fluctuations or drift. Actin filaments and microtubules should appear co-entangled and homogeneously distributed, with minimal bundling, aggregation, or phase separation of actin and microtubules throughout a field of view of ~200 µm x 200 µm (Figure 2, far left)17. One should expect a similar result for composites that contain myosin but are not exposed to 488 nm light (to deactivate the blebbistatin).
Upon incorporation of myosin and exposure to 488 nm light, the composites undergo contraction that is largely isotropic and similar for actin and microtubules, as seen in microscope images taken before and after myosin activity (Figure 2), as well as corresponding PIV flow fields for varying times during activity (Figure 3). To determine if the motion is ballistic, diffusive, subdiffusive, etc., the characteristic decorrelation time τ(q) determined from DDM is evaluated as a function of wave vector (i.e., reciprocal space). See as described in detail previously17,26,49. Figure 4 also demonstrates how to use DDM to characterize these composites. Power-law scaling τ(q)~1/vqβ, with β = 1, indicates ballistic motion with speed v. For reference, β = 2 represents diffusive dynamics with v being the diffusion coefficient. All active composites exhibit ballistic scaling (Figure 4A) with speeds that are tuned by the concentrations of actin and myosin (Figure 4B), and that can vary in time during activity, either accelerating or decelerating (Figure 4C,D).
Network restructuring and clustering, visible in Figure 2 and more evident for higher actin and myosin concentrations, can be characterized using SIA, as depicted in Figure 5, and described previously17,48,50. Briefly, a correlation length ξ, which is a measure of the characteristic size of features in an image, can be determined by fitting each spatial intensity autocorrelation curve g(r) to an exponential function of distance r between pixels. Larger g(r) peaks that persist for longer distances indicate larger structural features (i.e., bundling, clustering of the individual filaments). As shown in Figure 5, for higher actin fractions and myosin concentrations, significant restructuring and aggregation is reflected in the increase in ξ over time.
The viscoelastic properties and nonlinear mechanical response of the active composites can also be measured using optical tweezers microrheology (OTM). However, protocols and representative results for these experiments are outside the scope of this work. Interested readers are referred to previous works48,56 which thoroughly describe how to perform OTM measurements and the expected results.
Using the same program of experimental and analysis tools described above, the following section describes how the dynamics and structure change when kinesin motors and biotin-NA crosslinkers are incorporated into the composites (Figure 6, Figure 7, and Figure 8). Figure 6 shows representative confocal images of composites driven by either kinesin-only (K) or kinesin and myosin (K+M), with and without passive crosslinking (XL) of actin filaments or microtubules.
Incorporating kinesin into composites initially results in similar dynamics and restructuring as myosin-driven composites as seen in the top row of Figure 7 (Class 1). However, the dynamics typically transition to large-scale anisotropic flow (Figure 7 middle row, Class 2), acceleration, and deceleration (Figure 7 bottom row, Class 3). These characteristics couple with mesoscale clustering and aggregation after 5-30 min (Figure 6 and Figure 8B). PIV-generated flow fields and temporal color maps shown in Figure 7 depict examples of isotropic restructuring (Class 1, top panel), directed flow (Class 2, middle panels), and bi-directional acceleration (Class 3, bottom panels).
Speeds of actin and microtubules at varying time points during activity, determined via fits to τ(q) curves, illustrate acceleration followed by deceleration (Figure 8), which depends on crosslinking. As also shown in Figure 8, when both motor proteins are incorporated, the dynamics are actually slower than kinesin-only composites, and there is delayed onset of mesoscale flow. Myosin also supports more homogeneous interpenetration of actin and microtubule networks throughout the duration of activity, as well as less aggregation and restructuring. These effects can be seen in the images in Figure 6 and are quantified by the time-varying correlation lengths computed via SIA, which are generally smaller in the presence of myosin (Figure 8B).
Figure 1. Design and characterization of active actin-microtubule composites with multiple force-generating motors and passive crosslinkers. (A) Actin monomers and tubulin dimers are co-polymerized at molar concentrations cA and cT of 0.73-11.6 μM and molar fractions of actin ΦA = cA / (cA + cT) = 0, 0.25, 0.5, 0.75, and 1, to form co-entangled networks of actin filaments (green) and microtubules (red). Passive crosslinking is achieved using NA to link biotinylated actin filaments (Actin XL) or microtubules (MT XL) at crosslinker:protein molar ratios of RA = 0.01-0.08 and RMT = 0.001-0.01 for actin and microtubules, respectively. Myosin-II mini-filaments (purple) and kinesin clusters (orange), at concentrations of cM = 0.12 – 0.48 μM and cK = 0.2 – 0.7 μM, push and pull on the filaments to drive the composites out of steady-state. (B) Schematic of formulation space. Myosin II mini-filaments (M), kinesin clusters (K), or both motors (K+M) are incorporated into composites with no passive crosslinkers (No XL), actin-actin crosslinks (Actin XL), and microtubule-microtubule crosslinks (MT XL). All cartoons are not drawn to scale. Please click here to view a larger version of this figure.
Figure 2. Two-color confocal imaging of myosin-driven cytoskeleton composites with varying myosin concentrations cM and molar actin fractions ΦA. (A) 256 x 128 square-pixel (212 x 106 μm2) two-color confocal microscopy images show how composites of actin filaments (green) and microtubules (red) are rearranged via myosin motor activity. No kinesin motors or passive crosslinkers are present. In each panel, images taken at the beginning (left, before) and end (right, after) of the 45 min myosin activation (via illumination with 488 nm light to deactivate blebbistatin) are shown. Panels are ordered by increasing molar concentration of myosin (cM), going from left to right, and increasing molar fraction of actin (ΦA), going from top to bottom. The colors outlining each panel match the color coding used in Figure 4 and Figure 5. Scale bars are 50 μM. To best capture dynamics and structure for analysis, we use frame rates of 1-5 fps, ROIs with 50-250 µm sides, and time-series durations of 5-45 min, depending on the rate of contraction and rearrangement. Panels in which the before and after images look similar indicate minimal restructuring, as seen in the pink, magenta, and cyan panels. Small-scale clustering, evidenced by increased heterogeneity and the presence of bright punctate features, can be seen in the orange, green, and red panels. Large-scale contraction, seen as a uniformly shrinking network, is evident in the blue and purple panels. This figure has been modified from reference17. Please click here to view a larger version of this figure.
Figure 3. Particle image velocimetry (PIV) shows that actomyosin activity triggers coordinated contractile dynamics of actin and microtubules in co-entangled composites. PIV flow fields for actin (top row) and microtubules (bottom row) in a myosin-driven composite with (ΦA, cM) = (0.5, 0.24) at increasing times during a 6 min time-series. Flow fields were generated using the Fiji/ImageJ PIV plugin with a lag-time of 20 s and 2 pixel x 2 pixel binning. Both actin and microtubules show consistent motion directed towards the center region of the field-of-view throughout the movie duration. Scale bars in all images are 50 μm. Different arrow colors correspond to different speeds as indicated in the color scale to the right of vector fields. This figure has been modified from reference26. Please click here to view a larger version of this figure.
Figure 4. Time-resolved differential dynamic microscopy (DDM) measures the rate and type of motion of actin and microtubules in active composites. (A) DDM is performed on microtubule (top, open symbols) and actin (bottom, filled symbols) channels of time-series to determine characteristic decay times τ vs wavenumber q for both actin (filled symbols) and microtubules (open symbols) as described previously17,26. All curves follow τ ~ q-1 scaling, indicating ballistic motion, with speeds v that are determined via fits to τ(q) = (vq)-1. Faster speeds correspond to smaller τ(q) values for any given q. Symbol colors and shapes correspond to (ΦA, cM) combinations shown in B. (B) Contraction speeds v are determined via fits to τ(q) curves shown in A, which are averaged over all lag-times for the duration of each 45 min time-series. (C) Time-resolved DDM (trDDM) quantifies how the dynamics vary over time by evaluating τ(q) for actin (filled symbols, left) and microtubules (open symbols, right) for consecutive 6 min intervals (denoted by different shades of the same color) during the 45 min activation time. trDDM is performed for each (ΦA, cM) combination (denoted by different symbols and colors) as described in the legend in lower right. τ(q) curves shown in C follow similar scaling and trends as those in A but also show time-dependence for certain (ΦA, cM) compositions, most notably for ΦA = 0.75. (D) Contraction speeds for actin filaments (closed symbols) and microtubules (open symbols) are determined from fits to corresponding τ(q) curves. Error bars in all plots represent the standard error of values across three to five replicates. This figure has been modified from reference17. Please click here to view a larger version of this figure.
Figure 5. Spatial image autocorrelation (SIA) analysis quantifies the motor-driven restructuring of active cytoskeletal composites. (A) Autocorrelation g(r) for the microtubules at the beginning (left, t = 0 min, dark shades) and end (right, t = 42 min, light shades) of the experiment for (ΦA, cM) formulations listed in the legend. Inset: example fits of data to at the initial and final times for (ΦA, cM) = (0.75, 0.12). (B) Average correlation lengths ξ for actin (closed symbols) and microtubules (open symbols) for each (ΦA, cM) determined via exponential fits of each g(r) curve, as shown in the inset in A. Data is divided into those that exhibit minimal (left) versus substantial (right) restructuring. Error bars in A and B represent the standard error across three to five replicates. This figure has been modified from reference17. Please click here to view a larger version of this figure.
Figure 6. Incorporating kinesin motors and passive crosslinkers into active composites to increase programmability and expand the phase space of dynamics and structure. (A) Two-color confocal images of actin (green) and microtubules (red) in active composites show complex formulation-dependent restructuring over time (listed in min). The five images in each row correspond to five frames of a 2000 frame time-series acquired for a composite driven by kinesin (K, rows 1, 3, 5) or kinesin and myosin (K+M, rows 2, 4, 6), and including either no passive crosslinkers (No XL, rows 1, 2), actin-actin crosslinks (Actin XL, rows 3, 4), or microtubule-microtubule crosslinks (MT XL, rows 5, 6). Scale bars are all 50 µm. Outline colors match color scheme in Figure 8. (B) Separate actin and microtubule fluorescence channels for the kinesin-only composites show varied structures with both actin-MT co-localization and micro-phase separation. The images shown are for composites with cA = 2.32 μM, cT = 3.48 μM, cK = 0.35 μM, cM = 0.47 μM (rows 2, 4, 6), RA = 0.02 (rows 3, 4), and RMT = 0.005 (rows 5, 6). All composites begin with uniformly distributed interpenetrating networks of actin and microtubules (column 1). Kinesin-driven composites without crosslinkers (row 1) form loosely connected amorphous clusters that are MT-rich. Actin co-localizes in the centers of these aggregates initially but then is squeezed out of the MT-rich regions that continue to contract and disconnect from one another. Actin-actin crosslinking (row 3) hinders this microscale actin-MT separation, and instead MT-rich aggregates are connected via long strands of actin. Actin crosslinking also enables slow uptake of actin into the MT-rich regions, such that the composite becomes a connected network of co-localized actin and MT clusters. Microtubule crosslinking (row 5) leads to amorphous clustering of MTs that coalesce over time, resulting in larger scale phase separation of actin and MTs. Adding myosin (rows 2, 4, 6) reduces kinesin-driven de-mixing and restructuring. Without crosslinkers (row 2), composites show little rearrangement over the course of hours. Crosslinking increases restructuring and co-localization of actin and microtubules (rows 4, 6). Specifically, when microtubules are crosslinked (row 6), there is significant interpenetration and reorganization into web-like networks of fibers. This figure has been modified from reference50. Please click here to view a larger version of this figure.
Figure 7. PIV shows that active composites exhibit three classes of spatiotemporally distinct flow fields. (A) PIV flow fields for the first (ti) and last (tf) frames of three representative time-series, showing the different dynamical classes that composites shown in Figure 6 exhibit. PIV flow fields for microtubules (top) and actin (bottom) for class 1 (top, purple), class 2 (middle, orange), and class 3 (bottom, magenta) example videos, with arrow colors corresponding to the universal speed scale at the bottom, and the grayscale colormap showing the spatial speed distribution, normalized separately for each flow field according to the scale shown at bottom. Scale bars are all 50 μM. (B) Angular distributions of velocity vectors from A (in units of radians) with listed initial and final standard deviations σi and σf. (C) Temporal color maps for the videos analyzed in A and B show the frame-to-frame position of each pixel relative to its starting point. Class 1 maps show small-scale random motion; class 2 maps depict fast unidirectional motion with minimal spatial or temporal variation; class 3 maps exhibit features of both class 1 and 2. This figure has been modified from reference50. Please click here to view a larger version of this figure.
Figure 8. DDM and SIA measure the time-varying dynamics and structure of two-motor actin-microtubule composites. (A) Speeds for composites described in Figure 6 and Figure 7, measured via DDM, show acceleration and deceleration of composites, programmed by crosslinking and myosin activity. Speeds of microtubules (MT, closed circles) and actin (A, open circles) are plotted as a function of activity time in composites with no crosslinking (top, blue), actin crosslinking (middle, green), microtubule crosslinking (bottom, red), without myosin (K, darker shades), and with myosin (K+M, lighter shades). For class 3 cases, which have two speeds, the slower speed is indicated by a star. Data points enclosed by dashed black circles correspond to maximum speed vmax for each formulation. Error bars (most too small to see) are the standard error over the power-law fits of the corresponding τ(q). (B) Structural correlation lengths ξ, determined via SIA, versus activity time, for the same set of time-series evaluated in A. Each data point is an average of the correlation lengths determined for the first and last frame of the corresponding time-series. In general, ξ increases in time for both actin and microtubules in all composites systems, and composites driven solely by kinesin have greater correlation lengths than those in which myosin is also present. Data points in A and B that correspond to the three time-series analyzed in Figure 7 are circled in the corresponding class color (1 = purple, 2 = orange, 3 = magenta). This figure has been modified from reference50. Please click here to view a larger version of this figure.
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 μM)57,58,59 and tubulin (~3 – 4 μM)60, and at the upper limit by the transition to nematic alignment of actin filaments (~90 μM)61,62 or microtubules (~35 μM)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 °C in single-use aliquots. Once removed from -80 °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 °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 °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 °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.
The authors have nothing to disclose.
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.
(-)-Blebbistatin Abbreviation used in paper: blebbistatin |
Sigma Aldrich | B0560 | Stock Concentration: 200 μM in DMSO Storage: dessicated, in DMSO, -20ºC Stock and Experiment Recipes: dissolve 1 mg of powder to 200 μM in DMSO Storage, Handling, Troubleshooting Notes: limited shelf-life, typically stops functioning reliably after 3-4 months. purchase and prepare new solution every 3 months. |
1:20 488-tubulin:tubulin mixture Abbreviation used in paper: 5-488-tubulin |
NA | NA | Stock Concentration: 5 mg/ml in PEM Storage: single use aliquots, -80ºC, avoid freeze-thaw cycles Stock and Experiment Recipes: mix tubulin and 488-tubulin at a 20:1 ratio, flash freeze with LN2 Storage, Handling, Troubleshooting Notes: each aliquot can be used for up to 12 hrs stored on ice at 4ºC, protect from light |
1:20 R-tubulin:tubulin mixture Abbreviation used in paper: 5-R-tubulin |
NA | NA | Stock Concentration: 5 mg/ml in PEM Storage: single use aliquots, -80ºC, avoid freeze-thaw cycles Stock and Experiment Recipes: mix tubulin and rhodamine tubulin at a 20:1 ratio, flash freeze with LN2 Storage, Handling, Troubleshooting Notes: each aliquot can be used for up to 12 hrs stored on ice at 4ºC, protect from light |
actin (biotin): skeletal muscle Abbreviation used in paper: biotin-actin |
Cytoskeleton | AB07 | Stock Concentration: 1 mg/ml in G-buffer Storage: single use aliquots, -80ºC, avoid freeze-thaw cycles Stock and Experiment Recipes: reconstitute to 1 mg/ml in G-buffer, flash freeze with LN2 Storage, Handling, Troubleshooting Notes: (1) immediately prior to use dilute to 0.5 mg/ml in PEM, (2) once removed from -80ºC, store aliquot on ice at 4ºC for up to 1 week |
actin (rhodamine): rabbit skeletal muscle Abbreviation used in paper: R-actin |
Cytoskeleton | AR05 | Stock Concentration: 1.5 mg/ml in G-buffer Storage: single use aliquots, -80ºC, avoid freeze-thaw cycles Stock and Experiment Recipes: reconstitute to 1.5 mg/ml in G-buffer, flash freeze with LN2 Storage, Handling, Troubleshooting Notes: once removed from -80ºC, store aliquot on ice at 4ºC, can be used for up to 1 week |
adenosine triphosphate Abbreviation used in paper: ATP |
Thermo Fisher Scientific | A1048 | Stock Concentration: 100 mM Storage: in solution (pH 7), -20ºC Stock and Experiment Recipes: reconsitute in DI H20, bring pH to 7 with NaOH Storage, Handling, Troubleshooting Notes: routinely check pH and adjust as needed, hydrolyzes over time, replace every ~6-12 months |
AlexaFluor488 Phalloidin Abbreviation used in paper: 488-phalloidin |
Thermo Fisher Scientific | A12379 | Stock Concentration: 100 μM DMSO Storage: protected from light, dessicated, -20ºC Stock and Experiment Recipes: reconstitute to 100 μM with DMSO Storage, Handling, Troubleshooting Notes: immediately prior to use dilute to 20 μM in PEM (1 μL in 4 μL PEM) |
AlexaFluor488–labeled actin Abbreviation used in paper: 488-actin |
Thermo Fisher Scientific | A12373 | Stock Concentration: 1.5 mg/ml in G-buffer Storage: single use aliquots, -80ºC, avoid freeze-thaw cycles Stock and Experiment Recipes: reconstitute to 1.5 mg/ml in G-buffer, flash freeze with LN2 Storage, Handling, Troubleshooting Notes: this item has been discontinued |
Basic Plasma Cleaner Abbreviation used in paper: plasma cleaner |
Harrick Plasma | PDC-32G | |
Bemis Parafilm M Laboratory Wrapping Film Abbreviation used in paper: transparent film |
Thermo Fisher Scientific | 13-374-5 | |
D-(+)-Glucose Abbreviation used in paper: |
Thermo Fisher Scientific | A1682836 | Stock Concentration: 100x Storage: store at stock concentration (100x) or 10x concentration, dessicated, at -20ºC Stock and Experiment Recipes: reconstitute powder to 4.5 mg/ml in DI H20 Storage, Handling, Troubleshooting Notes: final concentration in solution should 45 μg/mL |
D-Biotin Abbreviation used in paper: biotin |
Fisher Scientific | BP232-1 | Stock Concentration: 1.02 mM in PEM Storage: dessicated, 4ºC |
deionized nanopure water Abbreviation used in paper: DI |
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Dimethyldichlorosilane Abbreviation used in paper: silane |
Thermo Fisher Scientific | D/3820/PB05 | Stock Concentration: 2% dissolved in Toulene |
Dithiothreitol Abbreviation used in paper: DTT |
Thermo Fisher Scientific | R0861 | Stock Concentration: 1 M in DMSO Storage: dessicated, -20ºC Stock and Experiment Recipes: dilute to 2 mM in PEM immediately before each experiment |
DMSO Anhydrous Abbreviation used in paper: DMSO |
Thermo Fisher Scientific | D12345 | |
F-Buffer Abbreviation used in paper: F-buffer |
NA | NA | Stock Concentration: 10x Storage: dessicated, -20ºC Stock and Experiment Recipes: 10 mM Imidazole (pH 7.0), 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 0.2 mM ATP |
G-Buffer Abbreviation used in paper: G-buffer |
NA | NA | Stock Concentration: 10x Storage: dessicated, -20ºC Stock and Experiment Recipes: 2.0 mM Tris (pH 8), 0.2 mM ATP, 0.5 mM DTT, 0.1 mM CaCl2. Store at -20°C. |
glass microscope slide Abbreviation used in paper: slide |
Thermo Fisher Scientific | 22-310397 | |
Glucose oxidase + catalase + β-mercaptoethanol Abbreviation used in paper: GOC |
Sigma Aldrich | G2133-250KU, C1345, 63689 | Stock Concentration: 100x Storage: store at stock concentration (100x) or 10x concentration, dessicated, at -20ºC Stock and Experiment Recipes: For 100x: 4.3 mg/ml glucose oxidase, 0.7 mg/ml catalase, 0.5% v/v β-mercaptoethanol in DI H20 Storage, Handling, Troubleshooting Notes: final concentration in solution should be: 0.005% β-mercaptoethanol, 43 μg/mL glucose oxidase, 7 μg/mL catalase |
glu-GOC oxygen scavenging system Abbreviation used in paper: glu-GOC |
NA | NA | Stock Concentration: 100x Storage: prepare fresh each time Stock and Experiment Recipes: mix equal parts Glu and GOC and add at 1/100 final sample volume immediately before imaging Storage, Handling, Troubleshooting Notes: prepare from Glu and GOC immediately before imaging |
Guanosine triphosphate Abbreviation used in paper: GTP |
Thermo Fisher Scientific | R0461 | Stock Concentration: 100 mM Storage: 100 μL aliquots at -20ºC |
Instant Mix 1-minute epoxy Abbreviation used in paper: epoxy |
Loctite | 1366072 | |
Kinesin-1 401 BIO 6x HIS Abbreviation used in paper: kinesin |
Prepared in JL Ross Lab at Syracuse University | NA | Stock Concentration: 8.87 μM in PEM Storage: single use aliquots, -80ºC, avoid freeze-thaw cycles Storage, Handling, Troubleshooting Notes: biotinylated dimers form kinesin clusters, each aliquot can be used for up to 12 hrs stored on ice at 4ºC |
NeutrAvidin Abbreviation used in paper: NA |
Thermo Fisher Scientific | 31000 | Stock Concentration: 5 mg/ml in PEM Storage: dessicated, -20ºC Stock and Experiment Recipes: reconstitute powder to 5 mg/ml in PEM |
No 1. glass coverslips (24 mm x 24 mm) Abbreviation used in paper: coverslip |
Thermo Fisher Scientific | 12-548-CP | |
Paclitaxel Abbreviation used in paper: Taxol |
Thermo Fisher Scientific | P3456 | Stock Concentration: 2 mM in DMSO Storage: protected from light, dessicated, -20ºC Stock and Experiment Recipes: reconstitute to 2 mM with DMSO Storage, Handling, Troubleshooting Notes: immediately prior to use dilute to 200 μM in DMSO (0.4 μL in 3.6 μL DMSO) |
PEM-100 Abbreviation used in paper: PEM |
NA | NA | Stock Concentration: 1x Storage: room temperature (RT) Stock and Experiment Recipes: 100 mM K-PIPES (pH 6.8), 2 mM EGTA, 2 mM MgCl2 Storage, Handling, Troubleshooting Notes: use KOH to adjust pH to 6.8, recheck pH often and adjust accordingly |
phalloidin Abbreviation used in paper: phalloidin |
Thermo Fisher Scientific | P3457 | Stock Concentration: 100 μM in DMSO Storage: protected from light, dessicated, -20ºC, adhere closely to storage/handling conditions Stock and Experiment Recipes: reconstitute to 100 μM with DMSO Storage, Handling, Troubleshooting Notes: susceptible to impurities in its preparation and denaturing, identifiable as large amorphous aggregates of actin in samples |
porcine brain tubulin Abbreviation used in paper: tubulin |
Cytoskeleton | T240 | Stock Concentration: 5 mg/ml in PEM Storage: single use aliquots, -80ºC, avoid freeze-thaw cycles Stock and Experiment Recipes: reconstitute powder to 5 mg/ml in PEM, flash freeze with LN2 Storage, Handling, Troubleshooting Notes: each aliquot can be used for up to 12 hrs stored on ice at 4ºC |
Potassium Chloride Abbreviation used in paper: KCl |
Thermo Fisher Scientific | AM9640G | Stock Concentration: 4 M Storage: RT |
Rabbit skeletal actin Abbreviation used in paper: actin |
Cytoskeleton | AKL99 | Stock Concentration: 2 mg/ml in G-buffer Storage: single use aliquots, -80ºC, avoid freeze-thaw cycles Stock and Experiment Recipes: reconstitute to 2 mg/ml in G-buffer, flash freeze with LN2 Storage, Handling, Troubleshooting Notes: once removed from -80ºC, store aliquot on ice at 4ºC, can be used for up to 1 week |
Rabbit skeletal myosin II Abbreviation used in paper: myosin |
Cytoskeleton | MY02 | Stock Concentration: 10 mg/ml in PEM Storage: single use aliquots, -80ºC, avoid freeze-thaw cycles Stock and Experiment Recipes: reconstitute powder to 10 mg/ml in PEM, flash freeze with LN2 Storage, Handling, Troubleshooting Notes: monomers form minifilaments at low KCl, each aliquot can be used for up to 12 hrs stored on ice at 4ºC |
Tubulin (biotin): porcine brain Abbreviation used in paper: biotin-tubulin |
Cytoskeleton | T333P | Stock Concentration: 5 mg/ml in PEM Storage: single use aliquots, -80ºC, avoid freeze-thaw cycles Stock and Experiment Recipes: reconstitute powder to 5 mg/ml in PEM, flash freeze with LN2 Storage, Handling, Troubleshooting Notes: immediately prior to use dilute to 0.5 mg/ml in PEM |
Tubulin (fluorescent HiLyte 488): porcine brain Abbreviation used in paper: 488-tubulin |
Cytoskeleton | TL488M | Stock Concentration: 5 mg/ml in PEM Storage: single use aliquots, -80ºC, avoid freeze-thaw cycles Stock and Experiment Recipes: reconstitute powder to 5 mg/ml in PEM, flash freeze with LN2 Storage, Handling, Troubleshooting Notes: each aliquot can be used for up to 12 hrs stored on ice at 4ºC, protect from light |
tubulin (rhodamine): porcine brain Abbreviation used in paper: R-tubulin |
Cytoskeleton | TL590M | Stock Concentration: 5 mg/ml in PEM Storage: single use aliquots, -80ºC, avoid freeze-thaw cycles Stock and Experiment Recipes: reconstitute powder to 5 mg/ml in PEM, flash freeze with LN2 Storage, Handling, Troubleshooting Notes: each aliquot can be used for up to 12 hrs stored on ice at 4ºC, protect from light |
Tween 20 Abbreviation used in paper: Tween20 |
Thermo Fisher Scientific | J20605.AP | Stock Concentration: 1% v/v in DI H20 Storage: RT |
ultracentrifuge grade microtubes Abbreviation used in paper: Beckman-Coulter Optima Max XP |
Beckman Coultier | 343776 | Storage, Handling, Troubleshooting Notes: 8×34 mm PC |
UV light curing glue Abbreviation used in paper: UV glue |
Pharda | SKG-2869 |