Spire1 and active INF2 promote ER-mitochondria contacts to enhance mitochondrial fission factor-mediated mitochondrial fission.

How interactions between the endoplasmic reticulum and mitochondria (ER-Mito) regulate mitochondrial fission remains a controversial topic. This study focuses on the molecular mechanism by which Spire type actin nucleation factor 1 (Spire1), mutant activator INF2 A149D (active INF2), and mitochondrial fission factor (MFF) regulate ER-Mito interactions to enhance mitochondrial fission. Our results show MFF-knockout severely inhibited mitochondrial fragmentation irrespective of Spire1 and active INF2 expression, demonstrating that MFF functions downstream of Spire1 and active INF2. Furthermore, we show a correlation between Spire1 and INF2 A149D expression and mitochondrial fission, confirming that an increase in ER-Mito contacts and mitochondrial fission events but a decrease in the duration of mitochondrial fission in the context of Spire1 and INF2 A149D overexpression. In addition, live-cell quantitative fluorescence resonance energy transfer (FRET) analysis found that the donor-centric FRET efficiency (ED)-concentration ratio of total acceptor-to-donor (RC) plots was lowered in the shSpire1 or shINF2 cells, indicating that Spire1 and INF2 may enhance the tightening of MFF oligomers to promote mitochondrial fission. Together our results show that Spire1 and active INF2 promote ER-mitochondria contacts to enhance MFF-mediated mitochondrial fission in MCF-7 cells.