How crosslinking actin filaments influences the microscale viscoelastic properties of actin-microtubule composites

Madison Francis1, Shea Ricketts1, Jennifer Ross2, Rae M Robertson-Anderson1

Department of Physics and Biophysics, University of San Diego1; Department of Physics, University of Massachusetts, Amherst1

The strength and mobility of cells is dependent upon the interactions between two protein filaments that comprise the cytoskeleton: actin and microtubules. These proteins form entangled networks that can also be chemically crosslinked to enable a wide range of mechanical properties. Here, we use optical tweezers microrheology to determine how varying concentrations of actin crosslinkers influences the viscoelastic properties of actin-microtubule composites. We create equimolar co-entangled networks of actin and microtubules with varying concentrations of actin crosslinkers. We use optical tweezers to apply both oscillatory and constant speed microscale strains over a range of rates and distances while simultaneously measuring the force the networks exert to resist these strains. We quantify the frequency-dependent complex viscosity, the nonlinear stress response, and the relaxation dynamics following strain. Surprisingly, we find that increasing the concentration of crosslinkers yields a decrease in network elasticity and stiffness.

*Funding: This work was funded by a NSF CAREER Award #1255446, a NIH NNIGMS Award #R15GM123420, and a W.M. Keck Foundation Research Grant.

Acknowledgements

We create equimolar composites of actin and microtubules with varying concentrations of actin crosslinkers

The force the composites exert to resist constant rate strains displays a surprising non-monotonic dependence on the actin crosslinker density

The force the composites retain following the strain displays the same surprising non-monotonic dependence on actin crosslinker density

As R increases from 0 to 0.01, the composite rigidity increases - as expected as entanglements are becoming permanently linked.However, increasing R beyond 0.01 yields a surprising decrease in the force response and rigidity of composites. The dependence of force on speed follows a power-law for all composites.

As R increases from 0 to 0.01, the composites exhibit less relaxation of force following strain and more mechano-memory.However, increasing R beyond 0.01 yields surprisingly faster relaxation behavior and more force dissipation.The dependence of retained force following relaxation on strain speed follows a power-law for all composites.

R=0

We quantify the density of crosslinking by the ratio of Neutravidin molecules to actin monomers ( R ).

R=0.08

We label a fraction of actin monomers with biotin to enable crosslinking by NeutrAvidin.

Oscillatory strain measurements show that the complex viscosity of composites displays the signature non-monotonic dependence on R

All composites exhibit shear thinning indicativeof viscoelastic entangled networks.

We apply small amplitude oscillatory strains tomeasure the complex viscosity of composites.We measure the amplitude F0 and phase shift Df ofthe force response relative to the oscillation of thebead position to determine the complex viscosity ofcomposites as function of frequency.

[NeutrAvidin]

[Actin]R=

We create composites byadding a custom-designedbuffer to an equimolarmixture of actin monomersand tubulin dimers. toimage composites, afraction of actin (green) andmicrotubules (red) arefluorescent-labeled.

The non-monotonic dependence offorce on R may indicate the emergenceof bundling and phase separation.

We use optically-trapped microspheres to apply strains of varying speeds and frequencies

We measure the force composites exert before, during and after the strain. We determine the force response and post-strain relaxation of force.

R=0.01 R=0.08

R=0.01

The average complex viscosity increases from R = 0 to R =0.01 then decreases. At R = 0.08 the viscosity is actuallylower than for composites with no linkers (R = 0).

The non-monotonic dependence of relaxationdynamics on R suggests the emergence ofincreasing composite mesh size from bundling.

R=0