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

Abstract

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 the 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 (also known as PTK2B), 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 <i>in situ</i>, 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.