Splitting Up: Finding a New Way to Monitor Mitochondrial CaMKII Using SplitGFP

Abstract

Important signaling events are specified by subcellular targeting, but current approaches may be inadequate to measure these events in living cells or in vivo. CaMKII (calcium/calmodulin-dependent protein kinase II) is an important mediator of cellular signaling, calcium homeostasis, heart failure, and lethal cardiac arrhythmias. There are two major CaMKII isoforms in the heart: γ and δ. Despite the broad functionality CaMKII has in regulating the cell, the availability of sensitive and specific tools to dynamically monitor CaMKII in subcellular compartments is limited. Recently, CaMKII was reported to reside in mitochondria where it mediates calcium homoeostasis and redox status in the organelle. Current methods of mitochondrial CaMKII detection rely on antibody based detection that cannot reliably distinguish between isoforms, and in some cases lack specificity. Here we developed a genetic reporter using SplitGFP to track mitochondrial CaMKII (mitoCaMKII) accumulation. Our strategy ‘splits’ self-complementing GFP, comprised of 11 β-strands, into two parts. First, GFP strands 1-10 were fused with a mitochondria localization sequence (MTS) and mCherry tag. Second, the GFP strand 11 was fused with CaMKIId. When both components are localized in the mitochondria the GFP self-complements and fluorescence reconstitutes. Using the excitable human retinal epithelial cell line (RPE1) we found that our CaMKIId-SplitGFP reporter is a sensitive marker of mitoCaMKII. Further, our reporter is capable of detecting dynamic increases of mitoCaMKII in response to β-adrenergic stress. These data suggest that CaMKII-SplitGFP is a useful tool in monitoring the dynamics and accumulation of mitoCaMKII, expanding the previously limited repertoire of CaMKII detection methods.