Acute diacylglycerol production activates critical membrane-shaping proteins leading to mitochondrial tubulation and fission.

Mitochondrial dynamics are orchestrated by protein assemblies that directly remodel membrane structure, however the influence of specific lipids on these processes remains poorly understood. Here, using an inducible heterodimerization system to selectively modulate the lipid composition of the outer mitochondrial membrane (OMM), we show that local production of diacylglycerol (DAG) directly leads to transient tubulation and rapid fragmentation of the mitochondrial network, which are mediated by isoforms of endophilin B (EndoB) and dynamin-related protein 1 (Drp1), respectively. Reconstitution experiments on cardiolipin-containing membrane templates mimicking the planar and constricted OMM topologies reveal that DAG facilitates the membrane binding and remodeling activities of both EndoB and Drp1, thereby independently potentiating membrane tubulation and fission events. EndoB and Drp1 do not directly interact with each other, suggesting that DAG production activates multiple pathways for membrane remodeling in parallel. Together, our data emphasizes the importance of OMM lipid composition in regulating mitochondrial dynamics.