auto-luminescent genetically-encoded ratiometric indicator for real-time ca2+ imaging at the single cell levelauto-luminescent基因编码的比率指标实时ca2 +成像在单细胞水平.pdf

Auto-Luminescent Genetically-Encoded Ratiometric Indicator for Real-Time Ca2+ Imaging at the Single Cell Level 1 1,2 1 1 3 Kenta Saito , Noriyuki Hatsugai , Kazuki Horikawa , Kentaro Kobayashi , Toru Matsu-ura , Katsuhiko Mikoshiba3, Takeharu Nagai1,4* 1 Research Institute for Electronic Science, Hokkaido University, Kita-20, Nishi-10 Kita-ku, Sapporo, Hokkaido, Japan, 2 Research Center for Cooperative Projects, Hokkaido University, Kita-15, Nishi-7 Kita-ku, Sapporo, Hokkaido, Japan, 3 Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako City, Saitama, Japan, 4 Precursory Research for Embryonic Science, Japan Science and Technology Agency, Sanbancho, Chiyoda-ku, Tokyo, Japan Abstract Background: Efficient bioluminescence resonance energy transfer (BRET) from a bioluminescent protein to a fluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaging in near real time without external light illumination. Methodology/Principal Findings: We applied BRET to develop an autoluminescent Ca2+ indicator, BRAC, which is composed of Ca2+-binding protein, calmodulin, and its target peptide, M13, sandwiched between a yellow fluorescent protein variant, Venus, and an enhanced Renilla luciferase, RLuc8. Adjusting the relative dipole orientation of the luminescent protein’s chromophores improved the dynamic range of BRET signal change in BRAC up to 60%, which is the largest dynamic range among BRET-based indicators reported so far. Using BRAC, we demonstrated successful