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

Goli Ardestani,Thomas J Maresca,Rafael A Fissore,Megan C West,Margaret M Stratton

doi:10.1074/jbc.ra119.009235

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.

Highlights

Ca2؉ oscillations and consequent Ca2؉/calmodulin-dependent protein kinase II (CaMKII) activation are required for embryogenesis, as well as neuronal, immunological, and cardiac signaling
We show CaMKII activity monitored in real-time following the induction of Ca2ϩ responses using several agonists that are capable of initiating embryogenesis
We show that FRESCA is reporting on endogenous CaMKII activity in the egg

Summary

Introduction

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. Unlike other Ca2ϩ/CaM-sensitive kinases, CaMKII acquires activity that is Ca2ϩ independent (autonomy) with sustained stimulation by autophosphorylation at Thr-286 (we will use CaMKII␣ numbering throughout the manuscript) (Fig. 1C) [12, 13]. As long as Thr-286 is phosphorylated, CaMKII will retain activity, even in the absence of Ca2ϩ It is this property, combined with its oligomeric organization, which provides the sensitivity of CaMKII to specific frequencies of Ca2ϩ rises

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