Cell-to-cell communication is critical to human physiology and diseases. In recent decades, the membrane enclosed, thin tube structures that directly connect cells, known as tunneling nanotubes (TNT), have gained recognition as an important mechanism of intercellular communication. Linear actin filament bundles are responsible the formation and function of TNTs. Various cellular materials can be transferred through these conduits, including protein aggregates, viruses, small molecules, ions and organelles. Consequently, TNTs contribute to multitude of human pathology propagations. Cancer cells can readily form nanotubes to redistribute chemotherapeutics and steal mitochondria from immune cells to evade treatments. Currently, molecular pathways and components that drive the formation and function of TNT are largely unknown. Our major goal of this proposal is to define key cytoskeletal mechanisms that govern cargo transport, structure and dynamics of TNTs. Our central hypothesis is that TNT linear actin is built through a specific set of actin binding proteins to facilitate actin-based content transfer between cells. We will employ quantitative single molecule localization microscopy, combined with cell and molecular biology methods to understand how actin within TNTs is spatiotemporally controlled to fulfill roles of both material transport within TNTs and sustain TNT structures. We will carry out two specific aims: (1) define the actin dynamics, directionality within TNTs, and (2) define key molecular players and mechanisms that control and maintain TNT architecture. The actin polarity, dynamics, and molecular motors will determine the mode of cargo transport. Identifying the parts-list associated with the TNT actin cytoskeleton will reveal the mechanisms of filament arrangement, maintenance, and assembly. Furthermore, expected findings are likely to implicate specific upstream signaling cascades in the formation of TNTs, enabling pharmacological intervention. The innovation of this project stems from tackling the protein chemistry of TNTs in living cells and at unprecedented spatiotemporal scale using cutting edge super-resolution technology. Additionally, the actin cytoskeleton is critical yet under-explored in the TNT system. This project will pave way to our long-term objective to provide potential targets to control TNT function in various disease contexts.