Ratiometric measurement of MAM Ca²⁺ 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 Ca²⁺ crosstalk. Despite the central importance of MAM Ca²⁺ dynamics in diverse biological processes, directly and specifically measuring Ca²⁺ concentrations inside MAM is technically challenging. Here, we develop MAM-Calflux, a MAM-specific BRET-based Ca²⁺ indicator. The successful application of the bimolecular fluorescence complementation (BiFC) concept highlights Ca²⁺-responsive BRET signals in MAM. The BiFC strategy imparts dual functionality as a Ca²⁺ indicator and quantitative structural marker specific for MAM. As a ratiometric Ca²⁺ indicator, MAM-Calflux estimates steady-state MAM Ca²⁺ levels. Finally, it enables the visualization of uneven intracellular distribution of MAM Ca²⁺ and the elucidation of abnormally accumulated MAM Ca²⁺ 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 Ca²⁺ communication.