A novel super-resolution STED microscopy analysis approach to observe spatial MCU and MICU1 distribution dynamics in cells

The uptake of Ca²⁺ by mitochondria is an important and tightly controlled process in various tissues. Even small changes in the key proteins involved in this process can lead to significant cellular dysfunction and, ultimately, cell death. In this study, we used stimulated emission depletion (STED) microscopy and developed an unbiased approach to monitor the sub-mitochondrial distribution and dynamics of the mitochondrial calcium uniporter (MCU) and mitochondrial calcium uptake 1 (MICU1) under resting and stimulated conditions. To visualize the inner mitochondrial membrane, the STED-optimized dye called pkMitoRed was used. The study presented herein builds on the previously verified exclusive localization of MICU1 in the intermembrane space, and that MCU moves exclusively laterally along the inner mitochondrial membrane (IMM). We applied a multi-angled arrow histogram to analyze the distribution of proteins within mitochondria, providing a one-dimensional view of protein localization along a defined distance. Combining this with optimal transport colocalization enabled us to further predict submitochondrial protein distribution. Results indicate that in HeLa cells Ca²⁺ elevation yielded MCU translocation from the cristae membrane (CM) to the inner boundary membrane (IBM). In AC16 cardiomyocyte cell line, MCU is mainly located at the IBM under resting conditions, and it translocates to the CM upon rising Ca²⁺. Our data describe a novel unbiased super-resolution image analysis approach. Our showcase sheds light on differences in spatial distribution dynamics of MCU in cell lines with different MICU1:MCU abundance.