Quantifying the inhibitory effect of Bcl-xl on the action of Mff using live-cell fluorescence imaging

Mitochondrial fission regulates mitochondrial function and morphology, and has been linked to Apoptosis. The mitochondrial fission factor (Mff), a tail-anchored membrane protein, induces excessive mitochondrial fission, contributing to mitochondrial dysfunction and Apoptosis. Here, we evaluated the inhibitory effect of Bcl-xL, an antiapoptotic protein, on the action of Mff by using live-cell fluorescence imaging. Microscopic imaging analysis showed that overexpression of Mff induced mitochondrial fragmentation and Apoptosis, which were reversed by coexpression of Bcl-xL. Microscopic imaging and live-cell fluorescence resonance energy transfer analysis demonstrated that Bcl-xL reconstructs the Mff network from punctate distribution of higher-order oligomers to filamentous distribution of lower-order oligomers. Live-cell fluorescence resonance energy transfer two-hybrid assay showed that Bcl-xL interacted with Mff to form heterogenous oligomers with 1 : 2 stoichiometry in cytoplasm and 1 : 1 stoichiometry on mitochondria, indicating that two Bcl-xL molecules primarily interact with four Mff molecules in cytoplasm, but with two Mff molecules on mitochondria.