Myosin-X Acts Upstream of L-Plastin to Drive Stress-Induced Tunneling Nanotubes

Tunneling nanotubes (TNTs) are thin, actin-based intercellular bridges that enable long-range communication during cellular stress; yet the molecular pathway controlling their formation remains unclear. Here, using gain- and loss-of-function approaches in Cath. a-differentiated (CAD) neuronal cells, we identified a unidirectional regulatory pathway in which myosin-X (Myo10) functions upstream of the actin-bundling protein L-(LCP1) to drive TNT formation. Using Western blotting and fluorescence microscopy, we determined that overexpression of L-plastin significantly increased the proportion of TNT-connected cells, whereas L-plastin downregulation reduced TNT formation, demonstrating that L-plastin is both sufficient and necessary for maintaining normal TNT abundance. Having previously shown that Myo10 is required for TNT formation in CAD cells, we asked whether the relationship is reciprocal. Overexpression/downregulation of L-plastin had no effect on Myo10 protein levels. Conversely, Myo10 downregulation decreased endogenous L-plastin by ~30%, and Myo10 overexpression elevated L-plastin expression and TNT number, demonstrating that Myo10 acts as an upstream regulator of L-plastin. Dual-color 3D imaging revealed co-localization of Myo10 and L-plastin along TNT shafts and filopodia-like precursors (Proto-TNTs). Together, these findings demonstrate that Myo10-dependent TNT formation requires the bundling protein L-plastin, providing a framework for how stress-induced signaling cascades couple TNT initiation to actin-core stabilization during stress and disease.