Ratiometric measurement of MAM Ca 2+ dynamics using a modified CalfluxVTN.

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.