Location-Dependent Photogeneration of Calcium Waves in HeLa Cells

Abstract

The calcium ion (Ca(2+)) concentrations in a cell are responsible for the control of vital cellular functions and have been widely studied as a means to investigate and control cell activities. Here, we demonstrate Ca(2+) wave generation in HeLa cells by femtosecond laser irradiation and show unexpected properties of the Ca(2+) release and propagation. When the laser was focused in the cell cytoplasm, Ca(2+) release was independent of both external Ca(2+) influx and the phosphoinositide-phospholipase C (PLC) signaling pathway. The nucleus was not a susceptible target for laser-induced Ca(2+) release, whereas irradiation of the plasma membrane produced evidence of transient poration, through which the extracellular solution could enter the cell. By chelating extracellular Ca(2+), we found that laser-induced influx of ethylene glycol tetra-acetic acid (EGTA) can compete with calcium induced calcium release and significantly delay or suppress the onset of the Ca(2+) wave in the target cell. Intercellular Ca(2+) propagation was adenosine triphosphate-dependent and could be observed even when the target cell cytosolic Ca(2+) rise was suppressed by influx of EGTA. The irradiation effect on overall cell viability was also tested and found to be low (85% at 6 h after irradiation by 60 mW average power). Laser-induced Ca(2+) waves can be reliably generated by controlling the exposure and focal position and do not require the presence of caged Ca(2+). The technique has the potential to replace other methods of Ca(2+) stimulation, which either require additional caged molecules in the cell or do not have an interaction that is as well localized.