Modulation of calcium signalling by intracellular organelles seen with targeted aequorins.

The cytosolic Ca(2+) signals that trigger cell responses occur either as localized domains of high Ca(2+) concentration or as propagating Ca(2+) waves. Cytoplasmic organelles, taking up or releasing Ca(2+) to the cytosol, shape the cytosolic signals. On the other hand, Ca(2+) concentration inside organelles is also important in physiology and pathophysiology. Comprehensive study of these matters requires to measure [Ca(2+)] inside organelles and at the relevant cytosolic domains. Aequorins, the best-known chemiluminescent Ca(2+) probes, are excellent for this end as they do not require stressing illumination, have a large dynamic range and a sharp Ca(2+)-dependence, can be targeted to the appropriate location and engineered to have the proper Ca(2+) affinity. Using this methodology, we have evidenced the existence in chromaffin cells of functional units composed by three closely interrelated elements: (1) plasma membrane Ca(2+) channels, (2) subplasmalemmal endoplasmic reticulum and (3) mitochondria. These Ca(2+)-signalling triads optimize Ca(2+) microdomains for secretion and prevent propagation of the Ca(2+) wave towards the cell core. Oscillatory cytosolic Ca(2+) signals originate also oscillations of mitochondrial Ca(2+) in several cell types. The nuclear envelope slows down the propagation of the Ca(2+) wave to the nucleus and filters high frequencies. On the other hand, inositol-trisphosphate may produce direct release of Ca(2+) to the nucleoplasm in GH(3) pituitary cells, thus providing mechanisms for selective nuclear signalling. Aequorins emitting at different wavelengths, prepared by fusion either with green or red fluorescent protein, permit simultaneous and independent monitorization of the Ca(2+) signals in different subcellular domains within the same cell.