Actin polymerization stimulates dynamics of both the inner and outer membrane during mitochondrial division Rajarshi Chakrabarti, Wei-Ke Ji, Radu Stan & Henry N. Higgs Department of Biochemistry and Cell Biology, Geisel School of Medicine, Dartmouth College, NH
Background:Successful mitochondrial division requires division of both the inner and outer mitochondrial membranes (IMM and OMM). OMM division depends upon the dynamin GTPase Drp1, which is preferentially recruited to ER-mitochondria contact sites. We have previously shown that actin polymerization by the ER-bound formin INF2 stimulates Drp1 mitochondrial recruitment and oligomerization. In this study, we asked whether INF2 played any role in IMM division. Results:We show that INF2-mediated actin polymerization plays a second role in mitochondrial division, stimulating IMM constriction through a calcium-dependent mechanism. INF2 activation causes a transient increase in mitochondrial matrix calcium, which dependens upon actin polymerization, myosin IIA activity, and ER calcium stores. Two findings suggest that the mechanism by which INF2 enhances mitochondrial calcium is by increasing ER/mitochondrial contacts: INF2 suppression eliminates an increase in stimulus-induced ER-mitochondrial contacts (distance <30 nm), and over-expression ER-mitochondrial tethers restores the stimulus-induced mitochondrial calcium increase in INF2-KO cells. Increased mitochondrial calcium correlates with synchronous mitochondrial constrictions of 1-2 m spacing. Knock-down of the mitochondrial calcium uniporter (MCU) strongly reduces the mitochondrial calcium increase, mitochondrial constrictions, and the mitochondrial division rate. Curiously, MCU-KD does not significantly change Drp1 oligomerization, and Drp1-KD causes an increase in mitochondrial constrictions, suggesting that these constrictions are Drp1-independent. Mitochondrial constrictions require the electron transport chain, but our current studies suggest they do not require the IMM dynamin GTPase Opa1. Live-cell Airyscan microscopy shows that IMM division occurs ~30 sec prior to OMM division. Conclusion and further results:These results demonstrate that actin polymerization through the ER-bound formin INF2 stimulates dynamics of both IMM and OMM during mitochondrial division. We also find that these INF2-mediated mitochondrial effects are distinct from actin “clouds” observed to cycle around mitochondria in mammalian culture cells.