**Rhodamine-labeled microtubules**

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 (*4-A*, *5-A* or *R-T*) to unlabeled (*A* or *T*) monomers can range from 1:50 to 1:5 depending on the overall protein concentration and type of fluorescent dye used. Below are recipes for optimized samples of entangled actin and actin-microtubule composites.

## *4.1.2.1 Example of in situ labeled actin network*

*c* = 1 mg/mL, [*5-A*]:[*A*] = 1:9.6, *VF* = 20 μL

6.3 μL PEM-100

#### **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.*

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*

[*T-P*] = 11.6 μM, *ϕ<sup>T</sup>* = 0.5, [*4-A*]:[*A*] = 1:4.8, [*R-T*]:[*T*] = 1:35.7, *VF* = 20 μL 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

**4.2 Fluorescence confocal microscopy methods**

*dependence of strain-induced actin mobility.*

mately reduces mobility.

with one another.

**207**

**Figure 5.**

single filaments within cytoskeleton composites can be resolved.

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

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

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

*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*

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 (*ϕ<sup>T</sup>* = 0.5). This surprising result, which aligns with the post-strain relaxation behavior (described in Section 3.3), arises from an interplay between varying mesh sizes and filament rigidity. Namely, as the fraction of microtubules in composites increases so does the mesh size, allowing for larger voids for filaments to move through (increasing mobility). However, increasing *ϕ<sup>T</sup>* eventually comes at a cost as the majority of filaments are rigid rods rather than semiflexible filaments, which hinders bending modes and fluctuations and ulti-

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

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.

#### *4.1.3 Labeling discrete filament segments for particle-tracking*

Because actin and microtubules are extended filaments, standard particletracking 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.

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 (*A*) and mix by pipetting 5 times. Incubate at RT for 20 min to allow labeled and unlabeled segments to anneal.

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*VF*, replacing the equivalent volume of PEM-100.

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

#### **Figure 5.**

1.2 μL 5-A 9.1 μL A

**Figure 4.**

0.4 μL 100 μM ATP 1 μL 1% Tween

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

2 μL oxygen scavenging system.

*Parasitology and Microbiology Research*

1 μL oxygen scavenging system

discrete-labeled actin filaments.

the equivalent volume of PEM-100.

filaments.

**206**

*4.1.2.2 Example of in situ labeled actin-microtubule composite*

*4.1.3 Labeling discrete filament segments for particle-tracking*

Shear filaments with a 26 gauge Hamilton syringe 15 times.

RT for 20 min to allow labeled and unlabeled segments to anneal. Prepare a 1:20 dilution in F-buffer. Mix by pipetting 5 times.

[*T-P*] = 11.6 μM, *ϕ<sup>T</sup>* = 0.5, [*4-A*]:[*A*] = 1:4.8, [*R-T*]:[*T*] = 1:35.7, *VF* = 20 μL

*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.*

Because actin and microtubules are extended filaments, standard particletracking 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

Follow protocol in Section 4.1.1.1 to prepare pre-formed Alexa-568-labeled actin

Quickly add 1 μL of 2 mg/mL actin (*A*) and mix by pipetting 5 times. Incubate at

Add to final sample solution from Section 2.2 at a volume of 0.05*VF*, replacing

*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.*

#### **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 (*ϕ<sup>T</sup>* = 0.5). This surprising result, which aligns with the post-strain relaxation behavior (described in Section 3.3), arises from an interplay between varying mesh sizes and filament rigidity. Namely, as the fraction of microtubules in composites increases so does the mesh size, allowing for larger voids for filaments to move through (increasing mobility). However, increasing *ϕ<sup>T</sup>* eventually comes at a cost as the majority of filaments are rigid rods rather than semiflexible filaments, which hinders bending modes and fluctuations and ultimately reduces mobility.

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].

**Acknowledgements**

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

Foundation Research Grant.

Authors declare no conflict of interest.

**Conflict of interest**

**Author details**

**209**

California, United States

Authors would like to thank Prof. Jennifer Ross (University of Massachussetts,

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

Amherst), Prof. Moumita Das (Rochester Institute of Technology), Robert

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

Shea N. Ricketts, Bekele Gurmessa and Rae M. Robertson-Anderson\* Department of Physics and Biophysics, University of San Diego, San Diego,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: randerson@sandiego.edu

provided the original work is properly cited.

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.
