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Acute Manipulation of Outer Membrane Phospholipid Composition Directly Alters Mitochondrial Dynamics and Ultrastructure

Mitochondria undergo coordinated rounds of fusion and fission that are critical for maintaining the functional integrity of this essential organelle. While a growing number of proteins have been identified as important regulators of mitochondrial dynamics, the direct role of membrane lipid composition during the fusion and fission processes is poorly understood. To address these shortcomings, we devised a protein‐engineering platform that allows for the acute remodeling of structural phospholipids within the outer mitochondrial membrane (OMM) of intact cells. Specifically, we modified a bacterial phospholipase C (Bacillus cereus (Bc)PI‐PLC) to initiate the rapid hydrolysis of phosphatidylinositol (PI) and locally generate diacylglycerol (DAG); an important intracellular signaling molecule and metabolic precursor that is used in diverse lipid biosynthetic pathways. Spatial restriction of enzyme activity was achieved using a chemically inducible system consisting of a rapamycin‐dependent dimerization module (FKBP‐BcPI‐PLC) along with an OMM targeting sequence tagged with the FKBP‐rapamycin binding domain (OMM‐FRB). Using these unique molecular tools, we show that recruitment of FKBP‐BcPI‐PLC to the OMM not only causes the expected local accumulation of DAG, but also initiates the rapid and uniform fragmentation of the mitochondrial network. Mitochondrial fission induced by FKBP‐BcPI‐PLC is accompanied by profound swelling of the mitochondrial matrix along with vesiculation of the inner mitochondrial membrane (IMM) and a general loss of cristae, which all occur within minutes of tethering FKBP‐BcPI‐PLC to the OMM. Expression of dominant‐negative constructs targeting essential GTPases known to regulate OMM fission suggest that both dynamin‐related protein 1 (Drp1) and dynamin 2 (Dnm2) work together to drive efficient BcPI‐PLC‐induced mitochondrial division. However, results using a validated Drp1 knockout cell line show that the loss of Drp1 alone is sufficient to prevent the mitochondrial fragmentation initiated by FKBP‐BcPI‐PLC recruitment, indicating that Drp1 likely functions upstream or independent of Dnm2 in this context. Interestingly, unlike the induced OMM fission, removal of Drp1 from cells does not prevent the matrix swelling or OMM constrictions observed in response to acute generation of DAG within the OMM. Ongoing experiments are now focused on characterizing new methods to sequentially metabolize the DAG generated within the OMM as well as investigate how local lipid composition influences the binding and oligomerization of membrane‐shaping proteins that may function in concert with Drp1 to regulate mitochondrial remodeling. Overall, these studies establish a direct relationship between lipid metabolism within the OMM and clinically relevant morphological changes that are known to manifest in mitochondrial‐associated diseases.