Ratiometric measurement of MAM Ca2+ dynamics using a modified CalfluxVTN
Eunbyul Cho, Young Sik Woo, Yeongjun Suh, Bo Kyoung Suh, Soo Jeong Kim, Truong Thi My Nhung, Jin Yeong Yoo, Tran Diem Nghi, Su Been Lee, Dong Jin Mun, Sang Ki Park
Abstract
Abstract Mitochondria-associated ER membrane (MAM) is a structure where these calcium-regulating organelles form close physical contact sites for efficient Ca 2+ crosstalk. Despite the central importance of MAM Ca 2+ dynamics in diverse biological processes, directly and specifically measuring Ca 2+ concentrations inside MAM is technically challenging. Here, we develop MAM-Calflux, a MAM-specific BRET-based Ca 2+ indicator. The successful application of the bimolecular fluorescence complementation (BiFC) concept highlights Ca 2+ -responsive BRET signals in MAM. The BiFC strategy imparts dual functionality as a Ca 2+ indicator and quantitative structural marker specific for MAM. As a ratiometric Ca 2+ indicator, MAM-Calflux estimates steady-state MAM Ca 2+ levels. Finally, it enables the visualization of uneven intracellular distribution of MAM Ca 2+ and the elucidation of abnormally accumulated MAM Ca 2+ from the neurons of Parkinson’s disease mouse model in both steady-state and stimulated conditions. Therefore, we propose that MAM-Calflux can be a versatile tool for ratiometrically measuring dynamic inter-organellar Ca 2+ communication.