FRET-based sensor for CaMKII activity (FRESCA): A useful tool for assessing CaMKII activity in response to Ca2+ oscillations in live cells

Abstract

Ca²⁺ oscillations and consequent Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) activation are required for embryogenesis, as well as neuronal, immunological, and cardiac signaling. Fertilization directly results in Ca²⁺ oscillations, but the resultant pattern of CaMKII activity remains largely unclear. To address this gap, we first employed the one existing biosensor for CaMKII activation. This sensor, Camui, comprises CaMKIIα and therefore solely reports on the activation of this CaMKII variant. Additionally, to detect the activity of all endogenous CaMKII variants simultaneously, we constructed a substrate-based sensor for CaMKII activity, FRESCA (FRET-based sensor for CaMKII activity). To examine the differential responses of the Camui and FRESCA sensors, we used several approaches to stimulate Ca²⁺ release in mouse eggs, including addition of phospholipase Cζ cRNA, which mimics natural fertilization. We found that the Camui response is delayed or terminates earlier than the FRESCA response. FRESCA enables assessment of endogenous CaMKII activity in real-time by both fertilization and artificial reagents, such as Sr²⁺, which also leads to CaMKII activation. FRESCA's broad utility will be important for optimizing artificial CaMKII activation for clinical use to manage infertility. Moreover, FRESCA provides a new view on CaMKII activity, and its application in additional biological systems may reveal new signaling paradigms in eggs, as well as in neurons, cardiomyocytes, immune cells, and other CaMKII-expressing cells.

Keywords: Ca2+/calmodulin-dependent protein kinase II (CaMKII); Camui; FRESCA (FRET-based sensor for CaMKII activity); biosensor; calcium imaging; fertility; fertilization; phosphorylation; reporter.