**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 [1–17]. 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.

**10 F-buffer**: 100 mM Imidazole (pH 7.0), 500 mM KCl, 10 mM MgCl2, 10 mM

**1% (v/v) Tween**: Used to prevent filaments from adsorbing to sample chamber

**100 mM GTP:** Store at 20°C. Dilute to experimental concentration in PEM-

**100 mM ATP (**pH to 7.0): Store at 20°C. Dilute to experimental concentration

**2 mM Taxol**: Suspend 1 mg Pacilitaxol (Sigma, T7402) in DMSO. Store at 20°C. **PEM-Taxol**: 198 μL PEM-100, 2 μL 2 mM Taxol. Make fresh for every sample.

**200 μM Taxol**: 18 μL DMSO, 2 μL 2 mM Taxol. Make fresh for every sample.

**Tubulin** (*T,* Cytoskeleton #T240): Resuspend to 5 mg/mL in PEM-100. Store in

**Biotinylated Tubulin** (*B-T*, Cytoskeleton #T333P): Resuspend to 5 mg/mL in

**Rhodamine Tubulin** (*R-T*, Cytoskeleton #TL590): Prepare 5 mg/mL solutions of 1:10 molar ratio [Rhodamine tubulin]:[tubulin]. Store in 5 μL aliquots at 80°C. *Example: bring 20 μg R-tubulin to 5 mg/mL by adding 4 μL PEM-100. Add 36 μL of*

**Actin** (*A*, Cytoskeleton, #AKL99): Resuspend lyophilized protein to 2 mg/mL in

**Biotinylated actin** (*B-A*, Cytoskeleton #AB07): Resuspend lyophilized protein

**Alexa-568-actin** (*5-A*, ThermoFisher #A12374): Dilute to 1.5 mg/mL in G-

**Alexa-488-actin** (*4-A*, ThermoFisher #A12373): Dilute to 1.5 mg/mL in G-

**Biotin** (*B*, Sigma #B4501): Resuspend to 102 mM in deionized water (DI) and

**NeutrAvidin** (*NA*, ThermoFisher #31000): Resuspend to 5 mg/mL in PEM-100.

**Experimental sample preparation:** For all cytoskeleton networks described below, a volume *VF* = 20 μL of protein monomers, reagents, and buffers are mixed together and quickly pipetted into a sample chamber constructed from a glass slide and a microscope coverslip separated by two layers of double-sided tape. Sample chambers are sealed with epoxy and incubated (time and temperature depend on

*2.2.1 Entangled actin at any concentration* c *(mg/mL) and final sample volume* VF

**Oxygen scavenging system**: 4.5 mg/mL glucose, 0.5% β-mercaptoethanol, 4.3 mg/mL glucose oxidase, 0.7 mg/mL catalase. Make fresh immediately prior to mixing into experimental sample. \**Used to slow photobleaching during imaging when*

*networks or microspheres have been fluorescent-labeled (see Section 4)*.

*Microscale Mechanics of Plug-and-Play In Vitro Cytoskeleton Networks*

in working buffer (PEM-100 or G-buffer) and keep on ice.

EGTA, 2 mM ATP. Store at 20°C.

*DOI: http://dx.doi.org/10.5772/intechopen.84401*

surface. Dilute in working buffer.

100 and keep on ice.

5 μL aliquots at 80°C.

PEM-100. Store in 2 μL aliquots at 80°C.

*5 mg/mL tubulin (T) to 4 μL R-tubulin*.

buffer. Store in 5 μL aliquots at 80°C.

buffer. Store in 5 μL aliquots at 80°C.

Store in 5 μL aliquots at 20°C.

VActin = (cVF)/[A]

**197**

VATP = 0.1VF 10 mM ATP VTween = 0.05VF 1% Tween

G-buffer. Store in 25 μL aliquots at 80°C.

to 1 mg/mL in G-buffer. Store in 5 μL aliquots at 80°C.

network) to form networks of filamentous proteins.

VPEM-100 = VF VActin VATP VTween VOS\*

**2.2 Entangled and crosslinked actin networks**

Store at RT.

Store at RT.

store at 4°C.

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, 18–20]. 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**

**PEM-100:** 100 mM K-PIPES (pH 6.8), 2 mM EGTA, 2 mM MgCl2. Store at room temperature (RT).

**G-buffer**: 2.0 mM Tris (pH 8), 0.2 mM ATP, 0.5 mM DTT, 0.1 mM CaCl2. Store at 20°C.

*Microscale Mechanics of Plug-and-Play In Vitro Cytoskeleton Networks DOI: http://dx.doi.org/10.5772/intechopen.84401*

**10 F-buffer**: 100 mM Imidazole (pH 7.0), 500 mM KCl, 10 mM MgCl2, 10 mM EGTA, 2 mM ATP. Store at 20°C.

**Oxygen scavenging system**: 4.5 mg/mL glucose, 0.5% β-mercaptoethanol, 4.3 mg/mL glucose oxidase, 0.7 mg/mL catalase. Make fresh immediately prior to mixing into experimental sample. \**Used to slow photobleaching during imaging when networks or microspheres have been fluorescent-labeled (see Section 4)*.

**1% (v/v) Tween**: Used to prevent filaments from adsorbing to sample chamber surface. Dilute in working buffer.

**100 mM GTP:** Store at 20°C. Dilute to experimental concentration in PEM-100 and keep on ice.

**100 mM ATP (**pH to 7.0): Store at 20°C. Dilute to experimental concentration in working buffer (PEM-100 or G-buffer) and keep on ice.

**2 mM Taxol**: Suspend 1 mg Pacilitaxol (Sigma, T7402) in DMSO. Store at 20°C. **PEM-Taxol**: 198 μL PEM-100, 2 μL 2 mM Taxol. Make fresh for every sample. Store at RT.

**200 μM Taxol**: 18 μL DMSO, 2 μL 2 mM Taxol. Make fresh for every sample. Store at RT.

**Tubulin** (*T,* Cytoskeleton #T240): Resuspend to 5 mg/mL in PEM-100. Store in 5 μL aliquots at 80°C.

**Biotinylated Tubulin** (*B-T*, Cytoskeleton #T333P): Resuspend to 5 mg/mL in PEM-100. Store in 2 μL aliquots at 80°C.

**Rhodamine Tubulin** (*R-T*, Cytoskeleton #TL590): Prepare 5 mg/mL solutions of 1:10 molar ratio [Rhodamine tubulin]:[tubulin]. Store in 5 μL aliquots at 80°C.

*Example: bring 20 μg R-tubulin to 5 mg/mL by adding 4 μL PEM-100. Add 36 μL of 5 mg/mL tubulin (T) to 4 μL R-tubulin*.

**Actin** (*A*, Cytoskeleton, #AKL99): Resuspend lyophilized protein to 2 mg/mL in G-buffer. Store in 25 μL aliquots at 80°C.

**Biotinylated actin** (*B-A*, Cytoskeleton #AB07): Resuspend lyophilized protein to 1 mg/mL in G-buffer. Store in 5 μL aliquots at 80°C.

**Alexa-568-actin** (*5-A*, ThermoFisher #A12374): Dilute to 1.5 mg/mL in Gbuffer. Store in 5 μL aliquots at 80°C.

**Alexa-488-actin** (*4-A*, ThermoFisher #A12373): Dilute to 1.5 mg/mL in Gbuffer. Store in 5 μL aliquots at 80°C.

**Biotin** (*B*, Sigma #B4501): Resuspend to 102 mM in deionized water (DI) and store at 4°C.

**NeutrAvidin** (*NA*, ThermoFisher #31000): Resuspend to 5 mg/mL in PEM-100. Store in 5 μL aliquots at 20°C.

**Experimental sample preparation:** For all cytoskeleton networks described below, a volume *VF* = 20 μL of protein monomers, reagents, and buffers are mixed together and quickly pipetted into a sample chamber constructed from a glass slide and a microscope coverslip separated by two layers of double-sided tape. Sample chambers are sealed with epoxy and incubated (time and temperature depend on network) to form networks of filamentous proteins.

#### **2.2 Entangled and crosslinked actin networks**

*2.2.1 Entangled actin at any concentration* c *(mg/mL) and final sample volume* VF

VPEM-100 = VF VActin VATP VTween VOS\* VActin = (cVF)/[A] VATP = 0.1VF 10 mM ATP VTween = 0.05VF 1% Tween

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,

**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, 18–20]. 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

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**

**PEM-100:** 100 mM K-PIPES (pH 6.8), 2 mM EGTA, 2 mM MgCl2. Store at room

**G-buffer**: 2.0 mM Tris (pH 8), 0.2 mM ATP, 0.5 mM DTT, 0.1 mM CaCl2. Store

mobility and structure (Section 4).

*Parasitology and Microbiology Research*

measurement and age of the sample.

temperature (RT).

at 20°C.

**196**

VOS = 0.05VF oxygen scavenging system\* \**If not imaging networks replace VOS with PEM-100*. Incubate at RT for 60 min.

Actin concentrations should be *c* = 0.1–2.5 mg/mL for entangled networks.

### *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 (*NA*), 2 biotins (*B*), and 2 biotin-actin monomers (*B-A*). The molar ratio *R* of crosslinker to total actin [*T-A*] can be varied according to the following:

[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 *X* in a volume *VFC*. Prepared complexes are viable for 24 h on ice.


VKCl = 2cMVF/[4 M KCl]

**Figure 1.**

**Figure 2.**

**199**

VTubulin = ϕT[T-P]VF/[T] VActin = (1 ϕT)[T-P]VF/[A] VGTP = 0.1VF 10 mM GTP

VOS = 0.05VFinal oxygen scavenging system\* \**If not imaging networks replace VOS with PEM-100*.

**2.4 Composite networks of actin and microtubules**

Co-entangled networks of actin and microtubules can be prepared with varying molar fractions of tubulin, *ϕ<sup>T</sup>* = [*tubulin*]/([*actin*]+[*tubulin*]), and total protein molarity, [*T-P*]=[*tubulin*]+[*actin*]. Composites are formed in PEM-100 with 1 mM ATP (for actin polymerization), 1 mM GTP (for tubulin polymerization) and 5 μM Taxol (for microtubule stabilization). To crosslink actin and/or microtubules within composites, biotin-NeutrAvidin complexes similar to those described in Section 2.2 can be prepared using either actin, tubulin, or both proteins (**Figure 2**).

*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.*

*Microscale Mechanics of Plug-and-Play In Vitro Cytoskeleton Networks*

*DOI: http://dx.doi.org/10.5772/intechopen.84401*

*2.4.1 Entangled actin-microtubule network with ϕ*T*, [T-P] and final sample volume VF*

VPEM-100 = VF VTubulin VActin VGTP VATP VTween VTaxol VOS\*

*(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).*

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 VCL V10xF VOS VActin = ([T-A]VF)/[A] VCL = VF/X V10 <sup>F</sup> = 0.1VF 10 F-buffer VOS = 0.05VFinal Oxygen scavenging system\* \**If not imaging networks replace VOS with G-buffer*.

#### **2.3 Reversibly bundled actin networks**

Bundled actin networks are formed via counterion condensation using high concentrations of MgCl2 and KCl. MgCl2 concentrations of *cM* > 4 mM will bundle actin when paired with KCl at a concentration of 2*cM* (**Figure 1**).

**Bundled actin network with actin concentration** *c* **(mg/mL) and MgCl2 concentration** *cM* **(mg/mL) in a final volume** *VF*

VPEM-100 = VF VActin VATP VTween VMgCl2 VKCl VOS\* VActin = cVF/[A] VATP = 0.1VF 10 mM ATP VTween = 0.05VF 1% Tween VMgCl2 = cMVF/[5 M MgCl2]

*Microscale Mechanics of Plug-and-Play In Vitro Cytoskeleton Networks DOI: http://dx.doi.org/10.5772/intechopen.84401*

#### **Figure 1.**

VOS = 0.05VF oxygen scavenging system\*

Incubate at RT for 60 min.

*Parasitology and Microbiology Research*

[B-A] = ½[B]

\**If not imaging networks replace VOS with PEM-100*.

*2.2.2 Pre-assembled biotin-NeutrAvidin crosslinker assay*

total actin [*T-A*] can be varied according to the following:

Sonicate complex solution for 90 min at 4°C.

*2.2.3 Crosslinked network with any given R and [T-A]*

VG-buffer = VF VActin VCL V10xF VOS

VOS = 0.05VFinal Oxygen scavenging system\* \**If not imaging networks replace VOS with G-buffer*.

**concentration** *cM* **(mg/mL) in a final volume** *VF*

Add volume VCL to solution below.

VActin = ([T-A]VF)/[A]

V10 <sup>F</sup> = 0.1VF 10 F-buffer

**2.3 Reversibly bundled actin networks**

VCL = VF/X

VActin = cVF/[A]

**198**

VATP = 0.1VF 10 mM ATP VTween = 0.05VF 1% Tween VMgCl2 = cMVF/[5 M MgCl2]

in a volume *VFC*. Prepared complexes are viable for 24 h on ice.

Actin concentrations should be *c* = 0.1–2.5 mg/mL for entangled networks.

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 (*NA*), 2 biotins (*B*), and 2 biotin-actin monomers (*B-A*). The molar ratio *R* of crosslinker to

[T-A] = [A] + [B-A]; R = [N-A]/([T-A]); R = ½ [B-A]/([T-A]); [NA] = ½

Concentration factor, X 2–20 4 μL G-buffer volume VG-Buffer = VFC VNA VB-A VB 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

Bundled actin networks are formed via counterion condensation using high concentrations of MgCl2 and KCl. MgCl2 concentrations of *cM* > 4 mM will bundle

**Bundled actin network with actin concentration** *c* **(mg/mL) and MgCl2**

actin when paired with KCl at a concentration of 2*cM* (**Figure 1**).

VPEM-100 = VF VActin VATP VTween VMgCl2 VKCl VOS\*

Recipe for preparing crosslinker complexes that are concentrated by a factor *X*

**Equations for a given R Ex. R = 0.07**

*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 timeseries (4 fps) taken on a Nikon A1R laser scanning confocal microscope with 60 objective.*

VKCl = 2cMVF/[4 M KCl] VOS = 0.05VFinal oxygen scavenging system\* \**If not imaging networks replace VOS with PEM-100*.

#### **2.4 Composite networks of actin and microtubules**

Co-entangled networks of actin and microtubules can be prepared with varying molar fractions of tubulin, *ϕ<sup>T</sup>* = [*tubulin*]/([*actin*]+[*tubulin*]), and total protein molarity, [*T-P*]=[*tubulin*]+[*actin*]. Composites are formed in PEM-100 with 1 mM ATP (for actin polymerization), 1 mM GTP (for tubulin polymerization) and 5 μM Taxol (for microtubule stabilization). To crosslink actin and/or microtubules within composites, biotin-NeutrAvidin complexes similar to those described in Section 2.2 can be prepared using either actin, tubulin, or both proteins (**Figure 2**).

*2.4.1 Entangled actin-microtubule network with ϕ*T*, [T-P] and final sample volume VF*

VPEM-100 = VF VTubulin VActin VGTP VATP VTween VTaxol VOS\* VTubulin = ϕT[T-P]VF/[T] VActin = (1 ϕT)[T-P]VF/[A] VGTP = 0.1VF 10 mM GTP

#### **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).*

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\* \**If not imaging networks replace VOS with PEM-100*. Incubate at 37°C for 60 min.
