title: Focal calcium monitoring with targeted nanosensors at the cytosolic side of endoplasmic reticulum.
abstract: 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.
authors.affiliation: Waseda Bioscience Research Institute in Singapore (WABIOS) , Singapore , Republic of Singapore.
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Hou
authors.affiliation: Waseda Bioscience Research Institute in Singapore (WABIOS) , Singapore , Republic of Singapore.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Arai
authors.affiliation: Department of Life Science & Medical Bioscience, Faculty of Science & Engineering, Waseda University , Tokyo , Japan.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Takei
authors.affiliation: Department of Life Science & Medical Bioscience, Faculty of Science & Engineering, Waseda University , Tokyo , Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Murata
authors.affiliation: Department of Life Science & Medical Bioscience, Faculty of Science & Engineering, Waseda University , Tokyo , Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Takeoka
authors.affiliation: Waseda Bioscience Research Institute in Singapore (WABIOS), Singapore, Republic of Singapore; Organization for University Research Initiatives, Waseda University, Tokyo, Japan; PRESTO, Japan Science and Technology Agency, Saitama, Japan.
authors.forename: Madoka
authors.initials: M
authors.lastname: Suzuki
keywords: 101 Self-assembly/Self-organized materials; 208 Sensors and actuators; 30 Bio-inspired and biomedical materials; Copper-free click reaction; N3-fura-2; calcium imaging; endoplasmic reticulum; nanosensor