Focal calcium monitoring with targeted nanosensors at the cytosolic side of endoplasmic reticulum

Ca²⁺ distribution is spatially and temporally non-uniform inside cells due to cellular compartmentalization. However, Ca²⁺ sensing with small organic dyes, such as fura-2 and fluo-4, has been practically applied at a single cell level where the averaged signal from freely diffusing dye molecules is acquired. In this study, we aimed to target azide-functionalized fura-2 (N₃-fura-2) to a specific site of subcellular compartments to realize focal Ca²⁺ sensing. Using scAVD (single-chain avidin)–biotin interaction and a copper-free click reaction system, we linked N₃-fura-2 to specifically-targeted scAVD protein fused with a red fluorescent protein mCherry, so that Ca²⁺ sensors conjugated with four N₃-fura-2 dyes with dibenzocyclooctyne (DBCO)-PEG4-biotin as a linker were generated at subcellular compartments in living cells. In cytoplasm, N₃-fura-2 showed a prolonged retention period after binding to scAVD. Furthermore, the reacted N₃-fura-2 was retained inside cells even after free dyes were washed out by methanol fixation. When scAVD was overexpressed on endoplasmic reticulum (ER) membranes, N₃-fura-2 was accumulated on ER membranes. Upon histamine stimulation, which increases cytosolic Ca²⁺ concentration, ER-localized N₃-fura-2 successfully sensed the Ca²⁺ level changes at the cytosolic side of ER membrane. Our study demonstrated specific targeting of N₃-fura-2 to subcellular compartments and the ability of sensing focal Ca²⁺ level changes with the specifically targeted Ca²⁺ sensors.