Dissecting cytoskeletal crosstalk through reconstituted actin–microtubule systems

Actin filaments and microtubules are fundamental components of the cytoskeleton in eukaryotic cells, orchestrating cellular processes such as intracellular transport, migration, and division. Traditionally studied as distinct entities, growing evidence highlights their intricate crosstalk, mediated by crosslinking proteins, motor proteins, and signaling pathways. Reconstituted in vitro systems provide a powerful framework for isolating and probing these interactions under controlled conditions, enabling direct connections between molecular coupling and higher-order cytoskeletal organization. Such approaches have revealed how actin-microtubule interactions shape network architecture, coordinate force transmission, and give rise to emergent mechanical behavior that cannot be inferred from either system alone. This review synthesizes mechanistic principles of actin-microtubule crosstalk revealed by reconstituted systems, spanning molecular interactions, network-scale organization, and mechanical feedback. These insights advance understanding of cytoskeletal coordination in cells and identify key challenges toward developing predictive frameworks that link molecular interactions to emergent cellular mechanics.