Mitochondrial fission is mediated by the highly conserved dynamin-related GTPase Drp1. Three mitochondrial membrane-anchored dynamin-related proteins, mitofusins (Mfn1 and Mfn2) that reside in the mitochondrial outer membrane (MOM) and OPA1 that is localized in the mitochondrial inner membrane (MIM), are responsible for fusion of the mitochondrial outer and inner membranes. The opposing processes of mitochondrial fission and fusion are mediated by several evolutionarily conserved large dynamin superfamily GTPases ( Pagliuso et al., 2018 Tilokani et al., 2018 Giacomello et al., 2020 Yu et al., 2020). Mitochondria are highly dynamic organelles that frequently change their shapes by shifting the balance between fission and fusion events in response to various cellular conditions. Collectively, our findings suggest that Mff and MIEFs respond differently to the molecular assembly state of Drp1 and that the extent of Drp1 oligomerization regulates mitochondrial dynamics. We here confirm that MIEFs also serve as a platform facilitating the binding of Drp1 to Mff and loss of MIEFs severely impairs the interaction between Drp1 and Mff. Moreover, all the fission-incompetent Drp1 mutants tested (except the monomeric mutant K668E) affect Drp1-driven mitochondrial dynamics via incorporation of the mutants into the native oligomers to form function-deficient Drp1 assemblies. Mff only recruits active forms of Drp1, while MIEFs are less selective and recruit both active and inactive Drp1 as well as oligomerization- or GTPase-deficient Drp1 mutants to mitochondria. Mff preferentially binds to higher order oligomers of Drp1, whereas MIEFs bind to a wider-range of Drp1 assembly subunits, including both lower and higher oligomeric states. Precluding mitochondria-bound Drp1 in Mff/MIEF1/2-deficient cells does not affect the oligomerization state of Drp1, while conversely forced recruitment of Drp1 to mitochondria by MIEFs or Mff facilitates Drp1 oligomerization. We show that in intact mammalian cells Drp1 exists as a mixture of multiple self-assembly forms ranging from the minimal, probably tetrameric, self-assembly subunit to several higher order oligomers. Here, we used in vivo chemical crosslinking to explore the self-assembly state of Drp1 and how it regulates the association of Drp1 with MIEFs and Mff. 3Department of Neurobiology, Care Sciences and Society, Center for Alzheimer Research, Division of Neurogeriatrics, Karolinska Institutet, BioClinicum, Solna, Swedenĭrp1 is a central player in mitochondrial fission and is recruited to mitochondria by Mff and MIEFs (MIEF1 and MIEF2), but little is known about how its assembly state affects Drp1 mitochondrial recruitment and fission.2Department of Cell and Molecular Biology, Karolinska Institutet, Biomedicum, Stockholm, Sweden.1Department of Oncology-Pathology, Karolinska Institutet, BioClinicum, Karolinska University Hospital Solna, Solna, Sweden.Rong Yu 1 Shao-Bo Jin 2 Maria Ankarcrona 3 Urban Lendahl 2,3 Monica Nistér 1* † Jian Zhao 1* †
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