The molecular architecture of tunneling nanotubes.

Tunneling nanotubes (TNTs) are thin intercellular bridges that mediate the exchange of proteins, organelles, and nucleic acids between neighboring cells. They are enriched in tumor cells, implicated in chemotherapy resistance, induced in models of aggregation-based diseases, and their formation is stimulated by viruses that use TNTs to enhance infection. Despite their broad relevance and therapeutic potential, TNT morphology and function remain poorly understood, owing to the absence of clear morphological criteria and the limitations of light microscopy. Here, we establish two complementary systems to study TNT formation and function: stimulation with the pseudorabies viral kinase US3 to model viral transmission, and treatment of acute monocytic leukemia THP-1 cells with daunorubicin to model chemotherapy resistance. Using live-cell imaging, we characterize cytoskeletal organization and bidirectional lysosome transport in both contexts, and apply cryo-correlative light and electron microscopy (cryo-CLEM) with cryogenic electron tomography (cryo-ET) to visualize TNTs in their native state at molecular resolution. We show that TNTs display a rich molecular architecture, comprising actin filaments, microtubules, intermediate filaments, active ribosomes, and diverse organelles including multivesicular bodies, autophagosomes, and lysosomes. Sub-nanometer microtubule reconstructions reveal mixed polarity within individual TNTs, suggesting that both connected cells actively contribute to TNT formation and cargo trafficking. This organization is conserved across both systems, implying that TNT biogenesis reflects a shared cellular program rather than a context-specific response. Our findings provide the first structural framework for TNTs, revealing an unexpectedly rich molecular architecture and laying the groundwork for understanding how TNTs orchestrate intercellular communication in disease.