Ca$^{2+}$-tunable mechanics and recoil in reconstituted Tcb2 networks

Tetrahymena calcium-binding protein (Tcb2) forms Ca$^{2+}$-responsive networks that exhibit contractile behavior, yet how Ca$^{2+}$ concentration controls their local mechanical response remains poorly understood. Here, we use optical tweezers to perform active microrheology on reconstituted Tcb2 networks inside a microfluidic device that enables precise control of Ca$^{2+}$ concentration, allowing systematic tuning of network structure and mechanics. We find that increasing Ca$^{2+}$ from 1 to 100~mM enhances the effective stiffness by nearly two orders of magnitude, from $\sim$1$\times$10$^{-3}$ to $\sim$7$\times$10$^{-2}$~pN/nm, corresponding to a transition from a viscosity-dominated to a more elastic and mechanically robust network. Recoil assays further reveal rapid release of stored elastic energy following deformation. After two orthogonal pulls, the bead recoils along the diagonal rather than retracing the loading path, indicating that stresses from different directions combine to produce a resultant restoring response. These results establish Tcb2 networks as a minimal, tunable system for probing chemomechanical coupling and viscoelasticity in Ca$^{2+}$-regulated protein networks.