Abstract
<jats:p> Tunneling nanotubes (TNTs) are actin-supported membrane bridges that mediate long-range intercellular communication and direct transfer of signaling molecules, organelles, and pathogenic cargo, yet the physicochemical mechanisms underlying their formation and organization remain poorly understood. Here we show that TNT-like intervesicular connections emerge from minimal physicochemical interactions between actin filaments and lipid membranes. Upon Mg <jats:sup>2+</jats:sup> exposure, actin-encapsulating vesicles spontaneously generated actin bundle-embedded lipid nanotubes (AT-LNTs) that formed stable intervesicular networks. Mg <jats:sup>2+</jats:sup> simultaneously induced actin polymerization, filament bundling, and electrostatic recruitment of F-actin to phosphatidylcholine membranes, enabling membrane tubulation without actin-binding proteins. Systematic perturbation of membrane phase, membrane tension, Mg <jats:sup>2+</jats:sup> concentration, ionic strength, and actin concentration revealed that AT-LNT formation occurs only within a narrow physicochemical regime where membrane deformation and actin–membrane coupling are simultaneously permissive. The resulting AT-LNTs reproduced key structural and dynamic features of cellular TNTs, including bundled organization, helical unwinding, lumenal diffusion, and spontaneous bridging between synthetic vesicles and living cells. These findings establish a minimal biophysical framework for understanding the emergence of intercellular membrane connections in living systems and provide a foundation for engineering communication between synthetic and living cells. </jats:p>