Communication of Ca2+ signals via tunneling membrane nanotubes is mediated by transmission of inositol trisphosphate through gap junctions

- Nanotubes are long, thin membranous connections between distant cells.
- Membrane nanotubes enable long-range Ca2+ signaling between distant cells.
- Functional coupling through gap junctions is required for this to occur.
- IP3 diffusion along the nanotube is responsible for Ca2+signal propagation.
- Ca2+ may be the 'message' but IP3 is the 'messenger'!

Tunneling membrane nanotubes (TNTs) are thin membrane projections linking cell bodies separated by many micrometers, which are proposed to mediate signaling and even transfer of cytosolic contents between distant cells. Several reports describe propagation of Ca2+ signals between distant cells via TNTs, but the underlying mechanisms remain poorly understood. Utilizing a HeLa M-Sec cell line engineered to upregulate TNTs we replicated previous findings that mechanical stimulation elicits robust cytosolic Ca2+ elevations that propagate to surrounding, physically separate cells. However, whereas this was previously interpreted to involve intercellular communication through TNTs, we found that Ca2+ signal propagation was abolished - even in TNT-connected cells - after blocking ATP-mediated paracrine signaling with a cocktail of extracellular inhibitors. To then establish whether gap junctions may enable cell-cell signaling via TNTs under these conditions, we expressed sfGFP-tagged connexin-43 (Cx43) in HeLa M-Sec cells. We observed robust communication of mechanically-evoked Ca2+ signals between distant but TNT-connected cells, but only when both cells expressed Cx43. Moreover, we also observed communication of Ca2+ signals evoked in one cell by local photorelease of inositol 1,4,5-trisphosphate (IP3). Ca2+ responses in connected cells began after long latencies at intracellular sites several microns from the TNT connection site, implicating intercellular transfer of IP3 and subsequent IP3-mediated Ca2+ liberation, and not Ca2+ itself, as the mediator between TNT-connected, Cx43-expressing cells. Our results emphasize the need to control for paracrine transmission in studies of cell-cell signaling via TNTs and indicate that, in this cell line, TNTs do not establish cytosolic continuity between connected cells but rather point to the crucial importance of connexins to enable communication of cytosolic Ca2+ signals via TNTs.

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