Imaging elemental events of store-operated Ca2+ entry in invading cancer cells with plasmalemmal targeted sensors

STIM1- and Orai1-mediated store-operated Ca2+ entry (SOCE) constitutes the major Ca2+ 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 Ca2+ signals. We visualized spontaneous discrete and long-lasting transients (“Ca2+ glows”) arising from STIM1-Orai1 in invading melanoma cells. Ca2+ glows occurred preferentially in single invadopodia and at sites near cell periphery under resting conditions. Re-addition of external Ca2+ after store depletion elicited spatially synchronous Ca2+ glows followed by high-rate discharge of asynchronous local events. Knockout of STIM1 or expression of the dominant-negative Orai1-E106A mutant markedly decreased Ca2+ glow frequency, diminished global SOCE and attenuated invadopodial formation. Functionally, invadopodial Ca2+ glows provided high Ca2+ microdomains to locally activate Ca2+/calmodulin-dependent Pyk2, which initiates the SOCE-Pyk2-Src signaling cascade required for invasion. Overall, the discovery of elemental Ca2+ signals of SOCE not only unveils a previously unappreciated gating mode of STIM1-Orai1 channels in situ, but underscores a critical role of the spatiotemporal dynamics of SOCE in orchestrating complex cell behaviors such as invasion.