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

Abstract

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.

Calcium sensing receptor protects high glucose-induced energy metabolism disorder via blocking gp78-ubiquitin proteasome pathway

“…11, 12 Impaired GJIC will result in the leakage of intracellular ATP. Therefore, the abnormal energy metabolism is not only related to the decrease of ATP synthesis, but also to the increase of ATP leakage.…”

Role of connexin 43 in different forms of intercellular communication – gap junctions, extracellular vesicles and tunnelling nanotubes

“…One of the main functions ascribed to TNTs is to mediate intercellular electrical coupling and Ca 2+ flux Watkins and Salter, 2005;Wittig et al, 2012). The finding that Cx43 is frequently present at one end of a TNT, together with the observation that electrical coupling between TNTconnected cells is inhibited in cells that lack gap junctions or when their function has been blocked, suggest that Cx43 is required for TNT-mediated electrical coupling (Lock et al, 2016;Smith et al, 2011;Wang et al, 2010;Wittig et al, 2012). In agreement, it has been shown that depolarization signals inflicted by mechanical stimulation can diffuse through Cx43-containing TNTs, which results in the activation of low-threshold voltage-gated Ca 2+ channels in non-stimulated cells .…”

“…In agreement, it has been shown that depolarization signals inflicted by mechanical stimulation can diffuse through Cx43-containing TNTs, which results in the activation of low-threshold voltage-gated Ca 2+ channels in non-stimulated cells . Moreover, it has been proposed that this signalling process relies on gap junctioncoupled TNT-mediated diffusion of inositol trisphosphate (IP 3 ) and low levels of Ca 2+ that can trigger Ca 2+ release from ER stores in the responding cell (Lock et al, 2016). Apart from Ca 2+ and IP 3 , it remains controversial which other molecules can spread through Cx43-containing TNTs.…”

Acetylcholine released by endothelial cells facilitates flow‐mediated dilatation

“…Instead, we used an alternative algorithm, developed by an independent group specifically for the automated detection and analysis of localized Ca 2+ signals in camera-based imaging data (Ellefsen et al 2014). This algorithm, and its use in assessing and characterizing localized Ca 2+ signalling in a number of cell types, has been demonstrated (Ellefsen et al 2014;Lock et al 2015Lock et al , 2016Schmunk et al 2015). Nevertheless, below we include a brief description of our use of the algorithm.…”