Location-dependent photogeneration of calcium waves in HeLa cells.
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.
Iwanaga
Shigeki
S
Department of Applied Physics, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Kaneko
Tomoyuki
T
Fujita
Katsumasa
K
Smith
Nicholas
N
Nakamura
Osamu
O
Takamatsu
Tetsuro
T
Kawata
Satoshi
S
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D004533:Egtazic Acid / Q000494:pharmacology; D006367:HeLa Cells / Q000166:cytology / Q000528:radiation effects; D006801:Humans; D007834:Lasers; D010716:Phosphatidylinositols / Q000378:metabolism; D010775:Photic Stimulation; D015398:Signal Transduction / Q000502:physiology; D010738:Type C Phospholipases / Q000378:metabolism
D002614:Chelating Agents; D010716:Phosphatidylinositols; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D010738:Type C Phospholipases; D002118:Calcium