Mitochondrial fission is essential for cellular homeostasis. Dysfunctional fission is implicated in neurodegenerative and cardiovascular diseases. In mammals, dynamin-related protein 1 (DRP1) drives fission by oligomerizing and constricting the mitochondrial outer membrane in a GTP-dependent manner. While DRP1 can bind to membranes itself, several recruiters facilitate binding, including the prominent mitochondrial fission factor (MFF). However, due to redundancies and the complex regulation of the rare fission process in vivo, the minimal fission machinery remains elusive. Here, we reconstituted mitochondrial fission using purified proteins and defined membrane compositions mimicking the outer mitochondrial membrane in an in vitro, high-contrast, fluorescence microscopy-based membrane tether assay supported by biochemical interaction and activity assays. To avoid DRP1-tagging artifacts, we used a fluorescent DRP1-specific nanobody. We found that single DRP1 complexes bound to tethers in either a stationary or diffusive manner and could constrict but not sever membrane tethers, independent of isoform. Only when MFF was bound, DRP1 complexes severed tethers with high efficiency. DRP1 and MFF isoforms and nucleotide states modulated oligomerization, membrane binding affinity, and the scission rate. In contrast to previous work, longer isoforms present in the brain were capable of fission, expanding our model toward neuronal-like conditions. In the long term, we expect to shed more light on what the minimal conditions and components are for mitochondrial fission and how the isoforms are adapted to the different cellular contexts.
Lurz et al. (Sun,) studied this question.