Tunneling nanotubes and epithelial bridges are recently discovered new forms of intercellular communication between remote cells allowing their electrical synchronization, transfer of second messengers and even membrane vesicles and organelles. In the present study, we demonstrate for the first time in primary cell cultures prepared from human laryngeal squamous cell carcinoma (LSCC) samples that these cells communicate with each other over long distances (up to 1 mm) through membranous tunneling tubes (TTs), which can be open-ended or contain functional gap junctions formed of connexin 43. We found two types of TTs, containing F-actin alone or F-actin and α-tubulin. In the LSCC cell culture, we identified 5 modes of TT formation and performed quantitative assessment of their electrical properties and permeability to fluorescent dyes of different molecular weight and charge. We show that TTs, containing F-actin and α-tubulin, transport mitochondria and accommodate small DAPI-positive vesicles suggesting possible transfer of genetic material through TTs. We confirmed this possibility by demonstrating that even TTs, containing gap junctions, were capable of transmitting double-stranded small interfering RNA. To support the idea that the phenomenon of TTs is not only typical of cell cultures, we have examined microsections of samples obtained from human LSCC tissues and identified intercellular structures similar to those found in the primary LSCC cell culture.

Gap junction (GJ) channels composed of Connexin36 (Cx36) are widely expressed in the mammalian CNS and form electrical synapses between neurons. Here we described a novel modulatory mechanism of Cx36 GJ channels that is dependent on intracellular free magnesium ([Mg2+]i). We examined junctional conductance (gj) and its dependence on transjunctional voltage (Vj) at different [Mg2+]i in cultures of HeLa or N2A cells expressing Cx36. We found that Cx36 GJs are partially inhibited at resting [Mg2+]i, thus, gj can be augmented or reduced by lowering or increasing [Mg2+]i, respectively. Similar changes in gj and Vj-gating were observed using MgATP or K2ATP in pipette solutions, which increases or decreases [Mg2+]i, respectively. Changes in phosphorylation of Cx36 or in [Ca2+]i were not involved in the observed Mg2+-dependent modulation of gj. Magnesium ions permeate the channel and transjunctional asymmetry in [Mg2+]i resulted in asymmetric Vj-gating. The gj of GJs formed of Cxs 26, 32, 43, 45 and 47 was also reduced by increasing [Mg2+]i, but was not increased by lowering [Mg2+]i; single channel conductance did not change. We showed that [Mg2+]i affects both open probability and the number of functional channels, likely through binding in the channel lumen. Finally, we showed that Cx36-containing electrical synapses between neurons of the trigeminal mesencephalic nucleus in rat brain slices are similarly affected by changes in [Mg2+]i. Thus, this novel modulatory mechanism could underlie changes in neuronal synchronization under conditions in which ATP levels, and consequently [Mg2+]i, are modified.

Key points• Under control conditions only one of ∼128 connexin (Cx)36 gap junction channels assembled in junctional plaques are open at a given time. This ratio was increased several times by short carbon chain n-alkanols and BSA, and reduced by long carbon chain n-alkanols.• Bovine serum albumin (BSA) increased junctional conductance (g j ) by removal of polyunsaturated fatty acids (PUFAs) including arachidonic acid (AA) from the plasma membrane of HeLaCx36-EGFP cells. BSA modified with 1,2-cyclohexanedione, which does not bind PUFAs, did not affect g j .• A primary culture of pancreatic β-cells, expressing solely Cx36, shows similar properties as HeLa transfectants, i.e. g j increases under an exposure to BSA and hexanol, while decanol and nonanol caused full uncoupling.• Methyl arachidonyl fluorophosphonate (MAFP) and thapsigargin, inhibitor and activator of AA synthesis, increased and reduced g j , respectively.• The g j -enhancing effect of hexanol did not change during combined application with MAFP and BSA, whereas AA and thapsigargin reduced the potentiating effect of hexanol.Abstract We examined junctional conductance (g j ) and its dependence on transjunctional voltage in gap junction (GJ) channels formed of wild-type connexin36 (Cx36) or its fusion form with green fluorescent protein (Cx36-EGFP) transfected in HeLa cells or endogenously expressed in primary culture of pancreatic β-cells. Only a very small fraction (∼0.8%) of Cx36-EGFP channels assembled into junctional plaques of GJs were open under control conditions. We found that short carbon chain n-alkanols (SCCAs) increased g j , while long carbon chain n-alkanols resulted in full uncoupling; cutoff is between heptanol and octanol. The fraction of functional channels and g j increased several fold under an exposure to SCCAs, or during reduction of endogenous levels of arachidonic acid (AA) by exposure to fatty acid-free BSA or cytosolic phospholipase A2 inhibitors. Moreover, uncoupling caused by exogenously applied AA can be rescued by BSA, which binds AA and other polyunsaturated fatty acids (PUFAs), but not by BSA modified with 1,2-cyclohexanedione, which does not bind AA and other PUFAs. We propose that under control conditions, Cx36 GJ channels in HeLa transfectants and β-cells are inhibited by endogenous AA, which stabilizes a closed conformational state of the channel that leads to extremely low fraction of functional channels. In addition, SCCAs increase g j by interfering with endogenous AA-dependent inhibition, increasing open probability and the fraction of functional channels.