Tunneling nanotubes (TNTs) are thin intercellular bridges that mediate communication and the exchange of proteins, organelles, and nucleotides between neighboring cells. They are enriched in tumor cells, linked to chemotherapy resistance, and readily form in models of aggregation-based genetic diseases, such as cystinosis and Parkinson’s disease. Viruses can also induce and hijack TNT-mediated transport to enhance infection. Despite their broad relevance and attractive therapeutic potential, TNT morphology and function remain controversial due to the absence of clear morphological criteria and the limitations of light microscopy. Here, we establish three complementary systems to study TNT formation and function. First, we stimulate TNTs with the pseudorabies viral kinase US3, mimicking viral transmission. Second, to model chemotherapy resistance, we treat monocytic leukemia THP-1 cells with daunorubicin. Third, to model genetic disease, we co-culture Sanfilippo type C fibroblasts, which carry a lysosomal storage disorder mutation, with IC21 macrophages. Using live-cell imaging, we track cytoskeletal organization and bidirectional lysosome transport in all three contexts. We then apply correlative cryo-electron tomography (cryo-ET) to visualize mature TNTs in their native environment. TNTs display a rich ultrastructure, including actin, microtubules, intermediate filaments, ribosomes, and diverse organelles such as multivesicular bodies, autophagosomes, and lysosomes. Subnanometer-resolution microtubule reconstructions reveal mixed polarity, suggesting contributions from both donor and acceptor cells to transport. Together, these results provide the first high-resolution structural framework of TNTs, revealing their molecular complexity and offering new insights into how TNTs may mediate intercellular communication across diverse biological contexts.
Karasmanis et al. (2026) studied this question.