The Role of different connexins in formation of tunneling nanotubes and transfer of small RNAs between HeLa cells

Abstract

Membranous intercellular tunneling tubes (TTs) are recently discovered new forms of communication between remote cells implicated in the cell migration; intercellular electrical coupling and Ca2+ flux; transfer of proteins, organelles and genetic material. In the present study, we employed the dual cell patch-clamp, fluorescence microscopy, time-lapse imaging and immunolabeling to examine the role of different connexins (Cx36, Cx40, Cx43, Cx45, and Cx47) expressed in HeLa cells in cell migration as well as formation and electrical and permeability properties of TTs. We demonstrate that HeLa cells expressing Cx36 exhibited the highest mobility properties compared with WT or other Cxs expressing HeLa cells. Thus, Cx36 which is highly expressed in nervous system and pancreas may be involved in controlling of cell migration and development of processes in these tissues. In WT or any Cx expressing HeLa cell culture, 2 types of TTs were identified – “thick” ones (from 300 nm up to 6.31 μm in diameter), containing F-actin and α-tubulin, and “thin” ones (diameter less than 300 nm), actin rich only. Thick TTs were formed during cytokinesis or by lamellipodium outgrowth mechanism, while thin ones were formed by filopodia extensions or protrusions. TTs established either open-ended or connexin-based, or close-ended connections between remote cells. TTs which were open-ended or connected the remote cells through Cx40, Cx43 or Cx47 GJs, were capable of transmitting while TTs containing Cx36 and Cx45 GJs were impermeable to the double-stranded small interfering RNA (siRNA/AF488). This newly described pathway of siRNA or miRNA direct delivery to the remote cells suggests the possible mechanism of cancer metastasis. Finally, we demonstrate that HeLa cells are capable of establishing the intercellular communication through TTs and Cx43 GJs with human mesenchymal stem cells which might be employed in gene silencing-based cancer therapy. This research was funded by