Functional visualization of the separate but interacting calcium stores sensitive to NAADP and cyclic ADP-ribose

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Abstract
Cells possess multiple Ca2+ stores and their selective mobilization provides the spatial-temporal Ca2+ signals crucial in regulating diverse cellular functions. Except for the inositol trisphosphate (IP3)-sensitive Ca2+ stores, the identities and the mechanisms of how these internal stores are mobilized are largely unknown. In this study, we describe two Ca2+ stores, one of which is regulated by cyclic ADP-ribose (cADPR) and the other by nicotinic acid adenine dinucleotide phosphate (NAADP). We took advantage of the large size of the sea urchin egg and stratified its organelles by centrifugation. Using photolysis to produce either uniform or localized increases of cADPR and NAADP from their respective caged analogs, the two separate stores could be visually identified by Ca2+ imaging and shown to be segregated to the opposite poles of the eggs. The cADPR-pole also contained the IP3-sensitive Ca2+ stores, the egg nucleus and the endoplasmic reticulum (ER); the latter was visualized using Bodipy-thapsigargin. On the other hand, the mitochondria, as visualized by rhodamine 123, were segregated to the opposite pole together with the NAADP-sensitive calcium stores. Fertilization of the stratified eggs elicited a Ca2+ wave starting at the cADPR-pole and propagating toward the NAADP-pole. These results provide the first direct and visual evidence that the NAADP-sensitive Ca2+ stores are novel and distinct from the ER. During fertilization, communicating signals appear to be transmitted from the ER to NAADP-sensitive Ca2+ stores, leading to their activation.