Excitation of mitochondria-Endoplasmic reticulum Ca2+ coupling by femtosecond-Laser photostimulation

Mitochondria locate close to endoplasmic reticulum (ER) and cooperate with ER to encode intracellular calcium signaling for cell processes and functions. However, the calcium coupling between mitochondria and ER (mito-ER) remains elusive due to the difficulty of precise excitation of localized mito-ER calcium coupling with little interference to other organelles. To study the mito-ER calcium coupling dynamics, we report a noninvasive method of photostimulation by femtosecond laser to excite the mito-ER calcium coupling at single-mitochondrion level. We establish a microscopy and photostimulation system to simultaneously stimulate and observe single mitochondrion continuously in real time, by which a long-term mito-ER calcium coupling is observed after a single short flash of femtosecond-laser stimulation to a tubular structure of a target mitochondrion in a pre-selected live cell. Cytosolic calcium is involved in the mito-ER calcium coupling but the mitochondrial sodium calcium exchanger and the mito-ER distance contribute little to it. Single mito-ER calcium coupling exhibits different dynamics from it excited by biochemical drugs globally. This study provides a precise and noninvasive method to excite calcium dynamics at single organelle level for the study of mitochondria and mito-ER calcium coupling.