Imaging elemental events of store-operated Ca entry in invading cancer cells with plasmalemmal targeted sensors.

STIM1- and Orai1-mediated store-operated Ca entry (SOCE) constitutes the major Ca influx in almost all electrically non-excitable cells. However, little is known about the spatiotemporal organization at the elementary level. Here, we developed Orai1-tethered or palmitoylated biosensor GCaMP6f to report subplasmalemmal Ca signals. We visualized spontaneous discrete and long-lasting transients ('Ca glows') arising from STIM1-Orai1 in invading melanoma cells. Ca glows occurred preferentially in single invadopodia and at sites near the cell periphery under resting conditions. Re-addition of external Ca after store depletion elicited spatially synchronous Ca glows, followed by high-rate discharge of asynchronous local events. Knockout of STIM1 or expression of the dominant-negative Orai1-E106A mutant markedly decreased Ca glow frequency, diminished global SOCE and attenuated invadopodial formation. Functionally, invadopodial Ca glows provided high Ca microdomains to locally activate Ca/calmodulin-dependent Pyk2 (also known as PTK2B), which initiates the SOCE-Pyk2-Src signaling cascade required for invasion. Overall, the discovery of elemental Ca signals of SOCE not only unveils a previously unappreciated gating mode of STIM1-Orai1 channels , but also underscores a critical role of the spatiotemporal dynamics of SOCE in orchestrating complex cell behaviors such as invasion. This article has an associated First Person interview with the first author of the paper.