Abstract
The coordination of microtubules (MTs) and actin filaments is essential for cytoskeletal organization, yet the factors that affect their integration remain unclear. Here, we reconstitute cytoskeletal networks in giant unilamellar vesicles to characterize MT-actin crosstalk mediated by tau, a microtubule-associated protein. We show that tau promotes the organization of MTs into diverse architectures, including bundles, clusters, and networks, depending on its concentration and vesicle size. In vitro assays confirm that while tau binds and bundles MTs, it does not directly bundle actin. However, tau facilitates MT-actin colocalization in the presence of actin crosslinkers with distinct properties. Fascin, which forms rigid actin bundles, significantly enhances MT-actin colocalization with tau, whereas α-actinin, which forms flexible actin bundles, induces colocalization in vesicles but not in bulk conditions. By combining cellular reconstitution and coarse-grained simulations of composite network assembly both in vesicles and in bulk conditions, our findings reveal how tau-mediated cytoskeletal integration is governed by bundle mechanics and spatial confinement, offering insights into cytoskeletal organization in both natural and synthetic cell-like systems.