Open access peer-reviewed chapter
Abstract
This chapter describes recent techniques that have been developed to reconstitute and characterize well-controlled, tunable networks of actin and microtubules outside of cells. It describes optical tweezers microrheology techniques to characterize the linear and nonlinear mechanics of these plug-and-play in vitro networks from the molecular-level to mesoscopic scales. It also details fluorescence microscopy and single-molecule tracking methods to determine macromolecular transport properties and stress propagation through cytoskeleton networks. Throughout the chapter the intriguing results that this body of work has revealed are highlighted—including how the macromolecular constituents of cytoskeleton networks map to their signature responses to stress or strain; and the elegant couplings between network structure, macromolecular mobility, and stress response that cytoskeleton networks exhibit.
Keywords
- cytoskeleton
- actin
- microtubules
- microrheology
- optical tweezers
- fluorescence
- microscopy
- in vitro
1. Introduction
The cell cytoskeleton is a complex and dynamic network of filamentous proteins that provides cells with structural and mechanical integrity while enabling key dynamic features such as cell motility, cytokinesis, apoptosis, and division [1, 2]. The cytoskeleton is able to perform these diverse functions by exhibiting a wide range of mechanical and structural properties that are tuned by the properties of, and interactions between, its constituent filamentous proteins: actin, microtubules, and intermediate filaments.
Due to the critical importance of understanding cytoskeleton mechanics and structure, over the past several decades numerous researchers from diverse disciplines have performed in vitro, in vivo, in silico, and theoretical studies aimed at elucidating this open problem [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17]. This collective body of work has made great strides in understanding the molecular structure and properties of individual actin filaments and microtubules, the viscoelastic properties of simple in vitro networks of cytoskeletal filaments, and the role that various crosslinking proteins play in the resulting architecture and mechanical properties of actin networks. Complementary in vivo studies have focused on identifying key motifs that arise in different cell types, in different regions of the cell, and during different phases in the cell cycle. These studies have demonstrated that the cytoskeleton can exhibit complex and nonlinear viscoelastic responses to strain; and that the lengths, concentrations and interactions between the comprising filaments play key roles in this response.
However, due to the diverse and complex mechanical responses and morphologies that cytoskeleton networks can exhibit, a connection between structure and mechanics in the cytoskeleton has proven elusive. The macromolecular properties and dynamics of the individual cytoskeleton filaments that give rise to the network stress response is also an open question. This chapter focuses on methods to overcome these issues including: the design of well-controlled in vitro cytoskeleton networks (Section 2), active microrheology methods to characterize the mechanical properties of these networks at the molecular and cellular scales (Section 3), and fluorescence microscopy techniques to measure network transport properties, mobility and structure (Section 4).
2. Preparation of tunable plug-and-play in vitro cytoskeleton networks
Over the past few decades, researchers have developed methods to create a range of cytoskeleton networks in vitro [3, 6, 11, 18, 19, 20]. Key issues that arise when creating and studying these networks are reproducibility and stability. There are also limited methods for creating networks comprised of multiple types of cytoskeleton filaments [6, 18]. Further, many of these systems exhibit structural and mechanical properties that vary from sample to sample, and exhibit aging and instability such that measurements are highly-dependent on the timescale of the measurement and age of the sample.
In vitro networks of semiflexible actin filaments have been most widely studied, spanning protein concentrations from the dilute to the nematic regimes, and incorporating numerous types of actin binding proteins (ABP) to create crosslinked and bundled networks [3, 13, 16, 19]. The motor protein, myosin II, has been used to create active and dynamic actin networks [4, 21, 22]. In vitro networks of rigid microtubules have also been studied, though less extensively [8, 14]. Far fewer studies have focused on composite networks of actin and microtubules, stemming from the incompatibility of established in vitro polymerization conditions for each protein. However, protocols have recently been developed to overcome this issue [18, 23].
Below are protocols to create highly stable, reproducible and tunable in vitro networks of actin and microtubules that mimic key biomimetic motifs and interactions. Further details regarding protocols can be found here [24]. These networks include actin networks with varying concentrations of crosslinkers, actin networks bundled by counterion condensation, and composite networks of sterically and chemically interacting actin filaments and microtubules. All networks are created by polymerization of actin monomers and/or tubulin dimers in an experimental sample chamber, rather than flowing in pre-formed filament networks, such that the native network structure and dynamics are preserved. To ensure reproducibility and stability, biotin-NeutrAvidin bonding and counterion condensation are used, rather than physiological ABPs, to create filament crosslinks and bundles.
2.1 Required buffers and reagents for networks described in Sections 2.2–2.4
2.2 Entangled and crosslinked actin networks
2.2.1 Entangled actin at any concentration c (mg/mL) and final sample volume V F
VPEM-100 = VF
VActin = (cVF)/[A]
VATP = 0.1VF 10 mM ATP
VTween = 0.05VF 1% Tween
VOS = 0.05VF oxygen scavenging system*
*
Incubate at RT for 60 min.
Actin concentrations should be
2.2.2 Pre-assembled biotin-NeutrAvidin crosslinker assay
To reproducibly form stable networks of crosslinked actin filaments that are isotropically crosslinked and free of bundling, it is important to pre-assemble Biotin-NeutrAvidin crosslinker complexes before adding to actin monomers to initiate network formation. Each complex is comprised of 1 NeutrAvidin (
[T-A] = [A] + [B-A]; R = [N-A]/([T-A]); R = ½ [B-A]/([T-A]); [NA] = ½ [B-A] = ½[B]
Recipe for preparing crosslinker complexes that are concentrated by a factor
| Equations for a given R | Ex. R = 0.07 | |
|---|---|---|
| Concentration factor, X | 2–20 | 4 μL |
| G-buffer volume | VG-Buffer = VFC | 2.4 μL |
| NeutrAvidin volume | VNA = X(VFCR[T-A]/[NA]) | 0.8 μL |
| Biotinylated actin volume | VBA = X(VFC2R[T-A]/[B-A]) | 5.6 μL |
| Biotin volume | VB = X(VFC2R[T-A]/[B]) | 1.2 μL |
Sonicate complex solution for 90 min at 4°C.
Add volume VCL to solution below.
2.2.3 Crosslinked network with any given R and [T-A]
VG-buffer = VF
VActin = ([T-A]VF)/[A]
VCL = VF/X
V10× F = 0.1VF 10× F-buffer
VOS = 0.05VFinal Oxygen scavenging system*
*
2.3 Reversibly bundled actin networks
Bundled actin networks are formed via counterion condensation using high concentrations of MgCl2 and KCl. MgCl2 concentrations of
Figure 1.
Confocal micrographs of actin networks (c = 5.8 μM) with varying degrees of bundling determined by the MgCl2 concentration (listed below each image). Images shown are average intensity projections from 60 s time-series (4 fps) taken on a Nikon A1R laser scanning confocal microscope with 60× objective.
VPEM-100 = VF
VActin = cVF/[A]
VATP = 0.1VF 10 mM ATP
VTween = 0.05VF 1% Tween
VMgCl2 = cMVF/[5 M MgCl2]
VKCl = 2cMVF/[4 M KCl]
VOS = 0.05VFinal oxygen scavenging system*
*
2.4 Composite networks of actin and microtubules
Co-entangled networks of actin and microtubules can be prepared with varying molar fractions of tubulin,
Figure 2.
(Left) Biotin-NeutrAvidin crosslinkers. (Right) Actin-microtubule networks in which: actin is crosslinked (Actin), microtubules are crosslinked (Microtubule), actin and microtubules are linked to each other (Co-linked), both the actin network and microtubule network are crosslinked (Both).
2.4.1 Entangled actin-microtubule network with ϕT , [T-P] and final sample volume VF
VPEM-100 = VF
VTubulin = ϕT[T-P]VF/[T]
VActin = (1
VGTP = 0.1VF 10 mM GTP
VATP = 0.1VF 10 mM ATP
VTween = 0.025VF 1% Tween
VTaxol = 0.025VF 200 μM Taxol (in DMSO)
VOS = 0.05VF oxygen scavenging system*
*
Incubate at 37°C for 60 min.
2.4.2 Recipe for preparing crosslinker complexes that are concentrated by a factor X in a volume VFC
Prepared complexes are viable for ∼24 h on ice. Biotinylated protein [
| Equations for a given R | Ex. R = 0.02 | |
|---|---|---|
| Concentration factor, X | Number ranging from 2 to 20 | 4 μL |
| PEM-100 volume | VPEM-100 = VFC | 5.28μL |
| NeutrAvidin volume | VNA = X(VFCR[T-P]/[NA]) | 2.79μL |
| Biotinylated protein volume | VBP = X(VFC2R[T-P]/[B-P]) | 1.02μL |
| Biotin volume | VB = X(VFC2R[T-P]/[B]) | 0.91μL |
Sonicate complex solution for 90 min at 4°C.
Add volume
2.4.3 Crosslinked network for any given R, ϕT and total protein concentration, [T-P]
VPEM-100 = VF
VTubulin = (ϕT[T-P]VF)/([T]
VActin = ((1-ϕT)[T-P]VF)/([A]
VCL = VF/X
VGTP = 0.1VF 10 mM GTP
VATP = 0.1VF 10 mM ATP
VTween = 0.025VF 1% Tween
VTaxol = 0.025VF 200 μM Taxol (in DMSO)
VOS = 0.05VF oxygen scavenging system*
*
3. Optical tweezers microrheology measurements
Given the importance of cytoskeleton mechanics to cell function, coupled with the complexity of mechanical properties that cells exhibit, understanding the response of cytoskeleton networks to stress and strain remains an important topic of research. Using standard bulk rheology techniques to measure the mechanical properties of cytoskeleton networks has been problematic due to the difficulty and expense in producing ∼mL sample volumes often needed for these measurements. Further, these measurements probe the macroscopic mechanical properties of the networks but are unable to probe mechanics at the molecular and cellular scales (∼μm). Finally, these methods are ill-equipped to measure spatial heterogeneities in network response, and can irreversibly disrupt or damage the network. Microrheology offers a complementary approach to characterizing the microscale mechanical and viscoelastic properties of cytoskeleton networks. While passive microrheology tracks freely diffusing microspheres embedded in networks to extract viscoelastic moduli, active microrheology uses optical tweezers to actively force embedded microspheres through networks and measure the force exerted to resist this strain. Active microrheology enables one to probe both molecular and mesoscopic scales and perturb networks far from equilibrium to access the nonlinear regime. Specifically, optical tweezers can be used to drag microspheres over distances that are large (5–30 μm) relative to the mesh size of the network (<μm) at speeds much faster than the molecular relaxation rates. The force exerted on the bead to resist the strain, as well as the subsequent relaxation of force following strain, is measured.
Reference [25] provides a thorough overview of the underlying principles and execution of optical tweezers microrheology to characterize the mechanics of biopolymer networks. Here, the focus is on the key results obtained using the in vitro cytoskeleton networks described in Section 2 [18, 19, 26, 27, 28].
3.1 Entangled actin networks
Active microrheology experiments have been carried out on entangled actin networks (Section 2.1) to characterize the dependence of the viscoelastic response and stress relaxation on the rate of the applied microbead strain
3.1.1 Strain rate dependence
Entangled actin networks (
3.1.2 Concentration dependence
These studies were extended to entangled actin networks of varying concentrations (
3.2 Crosslinked actin networks
As detailed in Section 2.2, methods have been developed to produce highly stable and reproducible networks of randomly-oriented crosslinked actin filaments. With these methods, the crosslinker density can be systematically tuned while fixing the actin concentration and structural network properties (i.e., isotropic filament orientation, no bundling).
Nonlinear microrheological characterization of these networks have been carried out for crosslinking ratios of
Following strain, all networks exhibit exponential force decay with two distinct timescales. Similar to the stress response characteristics, both fast and slow relaxation times scale exponentially with
These intriguing results, along with the corresponding actin filament deformations and stress propagation dynamics that lead to the force response, are further explored in Section 4.3.
3.3 Co-entangled composite networks of actin and microtubules
As described in Section 2.4, techniques have recently been developed to create randomly oriented, co-entangled networks of actin and microtubules by simultaneously co-polymerizing varying ratios of actin and tubulin in situ. The relative concentrations of actin and microtubules, quantified by the molar fraction of tubulin
Seminal microrheology studies on these composites have been carried out for
The presence of a large fraction of microtubules (
Force relaxation following strain exhibits two-phase power-law decay with the first decay arising from actin bending modes while the long-time relaxation is indicative of filaments reptating out of deformed entanglement constraints. Interestingly, the scaling exponents for the long-time relaxation exhibits a non-monotonic dependence on
4. Fluorescence imaging and characterization of network transport, mobility and structure
A key question regarding the cytoskeleton is how the mechanical force response couples to both network structure as well as the mobility and deformations of the comprising filaments. To address this problem, a range of fluorescence labeling schemes can be incorporated into in vitro networks, and various microscopy methods can be employed to image networks and quantify mobility and structure. This section describes different in vitro labeling and imaging methods as well as key results and parameters that can be obtained with the described methods.
4.1 Fluorescence labeling of proteins for varied measurement methods
Below are protocols for three different labeling schemes optimized for different network characterizations and imaging methods: (1) doping networks with pre-formed labeled filaments [18, 27], (2) in situ network labeling [23], and (3) labeling discrete filament segments for particle-tracking [19, 28].
4.1.1 Doping networks with pre-formed labeled filaments
This method is ideal for measuring filament length distributions and resolving single-filament fluctuations and mobility (Figure 3).
Figure 3.
Two-color laser scanning confocal imaging of fluorescent-labeled equimolar actin-microtubule composite ([T-P] = 11.6 μM). (A) The actin (green) channel, (B) microtubule (red) channel and (C) both channels show actin filaments and microtubules within composites form networks that overlap with each other forming a homogeneous network with no phase separation or clustering. The scale bar is 50 μm and applies to all images. The 512 × 512 image is taken on a Nikon A1R laser scanning confocal microscope with a 60× objective and QImaging CCD camera.
4.1.1.1 Labeled actin filaments for actin networks (Sections 2.2 and 2.3)
Prepare 10 μL of a 5 μM solution of 1:1 [
7.75 μL G-buffer
0.72 μL 5-A
0.54 μL A
1 μL 10× F-buffer
Incubate for 60 min at RT.
Prepare a 1:2 dilution in PEM-100 or F-buffer (depending on desired final buffer).
Add 1 μL of dilution to final sample chamber solution from Section 2.2 or 2.3, replacing the equivalent volume of PEM-100 or G-buffer (depending on network).
4.1.1.2 Labeled filaments for actin-microtubule composites (Section 2.4)
Prepare 10 μL of a 5 μM solution of 1:1 [
6.74 μL PEM-100
0.72 μL 4-A
0.54 μL A
2.00 μL 10 mM ATP
Incubate for 60 min at RT.
Immediately prior to imaging prepare a 1:2 dilution in PEM-100 + 2 mM ATP.
Add 1 μL to final sample chamber solution from Section 2.4, replacing the equivalent volume of PEM-100.
Prepare 5 μL of a 37 μM solution of R-T to polymerize prior to adding to composite as follows:
Thaw R-T aliquot in hand.
Add 0.55 μL 10 mM GTP.
Incubate at 37°C for 30 min.
Add 0.6 μL of 200 μM Taxol.
Incubate at 37°C for 30 min.
Immediately prior to imaging prepare a 1:10 dilution in PEM-Taxol.
Add 1 μL to final sample chamber solution from Section 2.4, replacing the equivalent volume of PEM-100.
Labeled filaments can be stored at RT for up to 1 week. After day 1, shear microtubules with a sterile hamilton syringe before adding to the sample chamber.
4.1.2 In situ network labeling
In this method labeled monomers are added to solution prior to in situ network formation, rather than adding pre-formed filaments [23]. This method, demonstrated in Figures 1 and 4 provides the most accurate depiction of network architecture and enables evaluation of network formation during polymerization. The drawback is that rarely are discrete single filaments visible, preventing filament length measurements. The ratio of labeled (
Figure 4.
Epifluorescence imaging of in situ labeling of an actin network (left), microtubule network (middle), and equimolar actin-microtubule composite (right). Images are sum projections of 400-frame time series (40 fps) taken using an Olympus IX73 microscope with 60× objective. The composite image also shows the separate channels for microtubules (top, far right) and actin (bottom, far right). All scale bars represent 10 μm.
4.1.2.1 Example of in situ labeled actin network
6.3 μL PEM-100
1.2 μL 5-A
9.1 μL A
0.4 μL 100 μM ATP
1 μL 1% Tween
2 μL oxygen scavenging system.
4.1.2.2 Example of in situ labeled actin-microtubule composite
[
3.43 μL PEM-100
0.29 μL 4-A
1.03 μL A
0.83 μL R-T
0.87 μL T
1 μL 10 mM ATP
1 μL 10 mM GTP
0.5 μL 200 μM Taxol
0.5 μL 1% Tween
1 μL oxygen scavenging system
4.1.3 Labeling discrete filament segments for particle-tracking
Because actin and microtubules are extended filaments, standard particle-tracking methods, optimized for punctile objects, cannot be used. To overcome this limitation, one can generate actin filaments with discrete, well-separated labeled segments (Figure 5) through a multi-stage polymerization process that includes shearing and annealing of labeled actin segments. Below is the protocol to create discrete-labeled actin filaments.
Figure 5.
Discrete labeling of actin filament segments for particle-tracking. (A) Microscope image of filament with interspersed ∼0.45 μm labeled segments that can be centroid-tracked during experiments. (B) Cartoon of tracking labeled segments along an actin filament. (C) Tracking discrete segments at varying distances R from strain path during microrheology experiments described in Section 3. Orange circles represent bead position before/after strain and yellow line represents strain path. Dotted lines outline annuli positioned every 4.5 μm from strain path (R = 6.75, 11.25, ..., 29.25 μm). All tracks within each annulus are used to determine R dependence of strain-induced actin mobility.
Follow protocol in Section 4.1.1.1 to prepare pre-formed Alexa-568-labeled actin filaments.
Shear filaments with a 26 gauge Hamilton syringe 15 times.
Quickly add 1 μL of 2 mg/mL actin (
Prepare a 1:20 dilution in F-buffer. Mix by pipetting 5 times.
Add to final sample solution from Section 2.2 at a volume of 0.05
4.2 Fluorescence confocal microscopy methods
Two-color fluorescence confocal microscopy allows for characterization of the 3D structure and mobility of networks comprised of multiple species (i.e., actin and microtubules). Use labeling method (1), the lengths, orientations, and mobility of single filaments within cytoskeleton composites can be resolved.
This method has been used to quantify the mobility of actin and microtubules within composites as described in Ref. [18]. Briefly, the standard deviation in pixel intensities over time can be computed from high frame-rate time-series and used to quantify the mobility of each filament type. Using this method, Reference [18] showed that the mobility of both actin and microtubules in co-entangled composites is greatest in equimolar composites (
3D stacks of images can also be used to determine network structure and connectivity. Evaluating these types of images has shown that the actin and microtubule networks comprising composites are isotropic, entangled, and well-integrated with one another.
More recently developed in situ labeling methods (Section 4.1.2) can more accurately depict network architecture [23] and can be analyzed to determine network correlation lengthscales and fluctuation rates.
4.3 Molecular-tracking microrheology
To directly track filament motion during and following strain, one can incorporate labeling technique (3) into cytoskeleton networks. This method results in punctile segments along filaments that can be tracked over time to determine filament trajectories. Incorporating fluorescence imaging and particle-tracking algorithms into an optical tweezers setup allows for imaging of these segments during micorheology measurements [19, 28].
This method has been used to couple filament deformations and strain propagation to force response in entangled and crosslinked networks of actin [19, 28]. One key result of this work was to determine the origin of stress stiffening and softening in crosslinked actin networks (see Section 3.2). In particular, these studies showed that initial stiffening arises from acceleration of strained filaments due to molecular extension along the strain path, while softening and yielding is coupled to filament deceleration, halting, and recoil. Networks also display a surprising non-monotonic dependence of filament deformation on crosslinker concentration. Namely, networks with no crosslinks or substantial crosslinks both exhibit fast initial filament velocities and reduced molecular recoil while intermediate crosslinker concentrations display reduced velocities and increased recoil. These collective results arise from a balance of network elasticity and force-induced crosslinker unbinding and rebinding. In accord with recent simulations [28], this work also showed that post-strain stress can be long-lived in crosslinked networks by distributing stress to a small fraction of highly strained connected filaments that span the network and sustain the load, while the rest of the network is able to recoil and relax.
5. Conclusions
As described in the preceding sections, several recent advances in in vitro network design and microrheological measurement techniques have enabled key insights into the mechanics and mobility of cytoskeleton networks.
The engineered networks include actin and microtubule networks with well-defined, versatile crosslinking motifs; networks of actin bundles mediated by counterion crossbridges, and composite networks of sterically and chemically interacting actin filaments and microtubules. The protocols and design schemes for these networks are highly modular to facilitate introducing higher levels of complexity and expanding the phase space of molecular constituents and structures. The versatile fluorescence labeling and imaging methods described allow for robust characterization of network dynamics and structure, while the active microrheology studies described can characterize the linear and nonlinear mechanical properties of these networks at the molecular and cellular scales. Some of the key findings this body of work has revealed include: the existence of critical strain rates and concentrations for actin networks to exhibit nonlinear mechanics, the inhomogeneous nature of stress propagation throughout crosslinked actin networks, the important role that actin plays in suppressing the buckling of microtubules, and the elegant competition between mesh size and polymer stiffness that leads to emergent dynamics in actin-microtubule networks.
While many open questions remain, these presented advances open the door for a wide range of highly-controlled new experiments to explore the vast phase space of mechanical and structural properties of diverse cytoskeleton networks.
Acknowledgments
Authors would like to thank Prof. Jennifer Ross (University of Massachussetts, Amherst), Prof. Moumita Das (Rochester Institute of Technology), Robert Fitzpatrick, Dr. Tobias Falzone, and Dr. Manas Khan for their contributions to the described work. This work was funded by an NSF CAREER Award (no. 1255446), an NIH-NIGMS Award (no. R15GM123420), Research Corporation & Gordon & Betty Moore Foundation Collaborative Innovation Award, and a W.M. Keck Foundation Research Grant.
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