INF 2-mediated actin polymerization at the ER stimulates mitochondrial calcium uptake , inner membrane constriction

Contacts between the ER and mitochondrion are now widely appreciated as important for communication in several respects, including lipid synthesis and calcium transfer (Phillips and Voeltz, 2016). For calcium transfer, close ER–mitochondrial contacts (<30 nm) enable a disproportionate amount of stimulus-induced calcium release from the ER to be taken up by mitochondria instead of being released into the cytosol (Rizzuto et al., 1998; Csordás et al., 2006, 2010; Giacomello et al., 2010). This uptake is mediated by the mitochondrial calcium uniporter (MCU; Baughman et al., 2011; De Stefani et al., 2011). Moderate increases in mitochondrial calcium stimulate oxidative catabolism through activation of several dehydrogenases (Denton, 2009), whereas excessive mitochondrial calcium can trigger apoptosis (Baffy et al., 1993). Although several proteins have been shown to mediate ER–mitochondrial contacts (Phillips and Voeltz, 2016), mechanisms controlling these contacts during cell stimulation are unclear. ER–mitochondrial contact also stimulates mitochondrial division (Friedman et al., 2011), which is required for diverse aspects of normal cellular physiology, including proper distribution of mitochondrial genomes (Lewis et al., 2016), metabolic adaptation (Mishra and Chan, 2016), mitophagy (Youle and van der Bliek, 2012; Burman et al., 2017), and immune response (Pernas and Scorrano, 2016). Defects in mitochondrial division have been linked to multiple pathologies, particularly neurodegenerative diseases (Nunnari and Suomalainen, 2012). Most mechanistic focus has been on outer mitochondrial membrane (OMM) division, with the dynamin GTPase Drp1 being a key factor (Labrousse et al., 1999; Labbé et al., 2014). Drp1 oligomerizes into a ring encircling the OMM, and GTP hydrolysis by Drp1 drives OMM constriction, leading to division. We have shown that one effector of ER-stimulated mitochondrial division in mammals is ER-bound inverted formin 2 (INF2), with INF2-mediated actin polymerization playing a key role in Drp1 recruitment to and oligomerization at division sites (Korobova et al., 2013; Ji et al., 2015). INF2 is linked to two human diseases: the neuropathy Charcot–Marie–Tooth disease (Boyer et al., 2011) and the kidney disease focal segmental glomerulosclerosis (Brown et al., 2010). Myosin II is also required for this process (DuBoff et al., 2012; Korobova et al., 2014), as well as the mitochondrially bound actin polymerization factor Spire1C (Manor et al., 2015). Other mechanisms of actin polymerization might also stimulate mitochondrial division (Li et al., 2015; Moore et al., 2016). Recent work shows that dynamin 2 accumulates on the OMM subsequent to Drp1 and acts at later stages of mitochondrial division (Lee et al., 2016). Comparatively little is known about inner mitochondrial membrane (IMM) division, which must also occur for successful mitochondrial division. Early results in Caenorhabditis Mitochondrial division requires division of both the inner and outer mitochondrial membranes (IMM and OMM, respectively). Interaction with endoplasmic reticulum (ER) promotes OMM division by recruitment of the dynamin Drp1, but effects on IMM division are not well characterized. We previously showed that actin polymerization through ER-bound inverted formin 2 (INF2) stimulates Drp1 recruitment in mammalian cells. Here, we show that INF2-mediated actin polymerization stimulates a second mitochondrial response independent of Drp1: a rise in mitochondrial matrix calcium through the mitochondrial calcium uniporter. ER stores supply the increased mitochondrial calcium, and the role of actin is to increase ER–mitochondria contact. Myosin IIA is also required for this mitochondrial calcium increase. Elevated mitochondrial calcium in turn activates IMM constriction in a Drp1-independent manner. IMM constriction requires electron transport chain activity. IMM division precedes OMM division. These results demonstrate that actin polymerization independently stimulates the dynamics of both membranes during mitochondrial division: IMM through increased matrix calcium, and OMM through Drp1 recruitment. INF2-mediated actin polymerization at the ER stimulates mitochondrial calcium uptake, inner membrane constriction, and division