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Ca2+ sent by tunneling membrane nanotubes is inositol trisphosphate-mediated
Members of the Smith Lab at the University of California, Irvine, have recently discovered that communication of Ca2+ signals via tunneling membrane nanotubes (TNT) is mediated by transmission of inositol trisphosphate (IP3) through gap junctions, at least in the HeLa M-Sec cell line. The Smith team began to work toward this finding after attempting to perform their original objective, which was to investigate previous reports that mechanical stimulation could invoke strong calcium responses that would propagate to surrounding cells, even if they were physically separate. With their findings published, they emphasize that there is a need to control for paracrine-mediated signaling, and intend to encourage more improved, reliable studies in TNT cell communication.
Since calcium signals were the main sign of cell communication the team chose to monitor throughout this study, calcium indicators Cal-520® and Cal-590™ were selected to visualize intracellular calcium concentrations for fluorescent calcium imaging. Cal-520® was first used to observe the rapid rise in calcium within a HeLa cell upon mechanical stimulation, which subsequently triggered other nearby cells to also express a calcium response, despite the absence of TNTs. To eliminate the suspected confounding factor of paracrine signaling causing such a phenomenon, ATP signaling blockers apyrase and suramin were added to the culture. However, the addition of these led to restriction of the calcium signal to the mechanically stimulated cell, even when TNTs connected them with neighboring cells. Thus far, the team decided to perform Cal-590™ calcium imaging on cells expressing connexins tagged with superfolded GFP (sfGFP). Cal-590™, a red-shifted dye, was utilized to allow for spectral separation between sfGFP and calcium images. Eventually the Smith team established that TNTs did not enable direct cytosolic contact in Hela cells, expression of connexins was required for transmissions of calcium signals by TNT, and long distance cell-cell communication of calcium happens through diffusion of IP3 along gap junction-coupled TNTs to induce calcium release from the ER of the responding cell.
Imaging all the calcium signals for the study and acquiring distinguishable readings with minimal background are enabled through calcium indicators Cal-520® and 590™. The Cal-series is best known for its notably enhanced intracellular retention, which reduces dye loss from the cytosol, and in turn ensures high signal-to-noise ratio and high sensitivity of its emitted signals. Improved retention also allows for cell incubation in the dye at low concentrations, as demonstrated by the 5 µM mentioned in the methods section of this study. Lower concentrations reduce sample exposure to harsh chemicals and minimize chances of unintentionally affecting calcium activity. Ultimately, Cal-520® and 590™ are ideal calcium indicators for accurate imaging of calcium dynamics within cells.
Since calcium signals were the main sign of cell communication the team chose to monitor throughout this study, calcium indicators Cal-520® and Cal-590™ were selected to visualize intracellular calcium concentrations for fluorescent calcium imaging. Cal-520® was first used to observe the rapid rise in calcium within a HeLa cell upon mechanical stimulation, which subsequently triggered other nearby cells to also express a calcium response, despite the absence of TNTs. To eliminate the suspected confounding factor of paracrine signaling causing such a phenomenon, ATP signaling blockers apyrase and suramin were added to the culture. However, the addition of these led to restriction of the calcium signal to the mechanically stimulated cell, even when TNTs connected them with neighboring cells. Thus far, the team decided to perform Cal-590™ calcium imaging on cells expressing connexins tagged with superfolded GFP (sfGFP). Cal-590™, a red-shifted dye, was utilized to allow for spectral separation between sfGFP and calcium images. Eventually the Smith team established that TNTs did not enable direct cytosolic contact in Hela cells, expression of connexins was required for transmissions of calcium signals by TNT, and long distance cell-cell communication of calcium happens through diffusion of IP3 along gap junction-coupled TNTs to induce calcium release from the ER of the responding cell.
Imaging all the calcium signals for the study and acquiring distinguishable readings with minimal background are enabled through calcium indicators Cal-520® and 590™. The Cal-series is best known for its notably enhanced intracellular retention, which reduces dye loss from the cytosol, and in turn ensures high signal-to-noise ratio and high sensitivity of its emitted signals. Improved retention also allows for cell incubation in the dye at low concentrations, as demonstrated by the 5 µM mentioned in the methods section of this study. Lower concentrations reduce sample exposure to harsh chemicals and minimize chances of unintentionally affecting calcium activity. Ultimately, Cal-520® and 590™ are ideal calcium indicators for accurate imaging of calcium dynamics within cells.
References
- Jeffrey T. Lock, Ian Parker, Ian F. Smith, Communication of Ca2+ signals via tunneling membrane nanotubes is mediated by transmission of inositol trisphosphate through gap junctions, Cell Calcium http://dx.doi.org/10.1016/j.ceca.2016.06.004
- Cal-520®, AM. AAT Bioquest, n.d. Web. 12 July 2016
- Cal-590™, AM. AAT Bioquest, n.d. Web. 12 July 2016
Original created on December 19, 2016, last updated on October 20, 2022
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