**Acknowledgements**

**4.3 Molecular-tracking microrheology**

*Parasitology and Microbiology Research*

during micorheology measurements [19, 28].

rest of the network is able to recoil and relax.

**5. Conclusions**

actin-microtubule networks.

**208**

To directly track filament motion during and following strain, one can incorpo-

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

As described in the preceding sections, several recent advances in in vitro network design and microrheological measurement techniques have enabled key

The engineered networks include actin and microtubule networks with welldefined, 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

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.

insights into the mechanics and mobility of cytoskeleton networks.

rate 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

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.
