Tunneling nanotubes (TNTs) are thin membranous channels providing a direct cytoplasmic connection between remote cells. They are commonly observed in different cell cultures and increasing evidence supports their role in intercellular communication, and pathogen and amyloid protein transfer. However, the study of TNTs presents several pitfalls (e.g., difficulty in preserving such delicate structures, possible confusion with other protrusions, structural and functional heterogeneity, etc.) and therefore requires thoroughly designed approaches. The methods described in this protocol represent a guideline for the characterization of TNTs (or TNT-like structures) in cell culture. Specifically, optimized protocols to (1) identify TNTs and the cytoskeletal elements present inside them; (2) evaluate TNT frequency in cell culture; (3) unambiguously distinguish them from other cellular connections or protrusions; (4) monitor their formation in living cells; (5) characterize TNTs by a micropatterning approach; and (6) investigate TNT ultrastructure by cryo-EM are provided. Finally, this article describes how to assess TNT-mediated cell-to-cell transfer of cellular components, which is a fundamental criterion for identifying functional TNTs. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Identification of tunneling nanotubes Alternate Protocol 1: Identifying the cytoskeletal elements present in tunneling nanotubes Alternate Protocol 2: Distinguishing tunneling nanotubes from intercellular bridges formed during cell division Basic Protocol 2: Deciphering tunneling nanotube formation and lifetime by live fluorescent microscopy Alternate Protocol 3: Deciphering tunneling nanotube formation using a live-compatible dye Basic Protocol 3: Assessing tunneling nanotubes functionality in intercellular transfer Alternate Protocol 4: Flow cytometry approach to quantify the rate of vesicle or mitochondria transfer Support Protocol: Controls to support TNT-mediated transfer Basic Protocol 4: Studies of tunneling nanotubes by cell micropatterning Basic Protocol 5: Characterization of the ultrastructure of tunneling nanotubes by cryo-EM.
Keywords: correlative cryo-electron microscopy (cryo-EM); cytoskeleton; intercellular communication; micropatterning; tunneling nanotubes (TNTs).
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