Mechanism of inverted formin 2 (INF2)-mediated effects on ER and mitochondria

• Applicant: Dr. Frieda Kage
• Subject Area: Cell Biology
• Term: from 2019 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 418076373

Mitochondria possess a characteristic morphology of remarkably high complexity. For maintenance of mitochondrial function, control processes such as fusion and fission are essential. One key component is the GTPase Drp1, which oligomerizes around and constricts the mitochondrion. Fission is known to preferentially occur at ER-mitochondrial contact sites, but the mechanism by which the ER stimulates mitochondrial fission is unclear. Recent results show that an ER-bound actin assembly factor, INF2, is important for mitochondrial fission. INF2-polymerized actin filaments stimulate mitochondrial fission in two ways: 1) by increasing Drp1 recruitment to mitochondrial fission sites; and 2) by stimulating ER-to-mitochondrial calcium transfer, with the increased mitochondrial calcium causing contractions of the inner mitochondrial membrane. My project addresses key unanswered questions in this developing mechanistic model. First, what population(s) of actin filaments are key to mitochondrial fission? INF2 causes assembly of at least three classes of polymerized actin: filaments at the fission site itself, filaments that run along the mitochondrion, and filaments in the bulk cytosol. I will use fluorescently-tagged CRISPR knock-in models to develop specific probes for localizing INF2 and actin populations by live-cell microscopy. Second, what is the precise role of INF2´s ER-localization in the context of mitochondrial fission? To address this question, I will follow two approaches: specific elimination of the ER-associated CAAX isoform and artificial targeting of INF2 to other organelles. Third, what role does myosin II play in mitochondrial fission? Previous work has shown that myosin II is required for both outer and inner mitochondrial membrane dynamics in mitochondrial fission, but its precise relationship with INF2-polymerized actin is unknown. Furthermore, it is unclear which of the three non-muscle myosin II proteins (myosin IIA, B or C) is/are important. I will develop knock-out and fluorescent knock-in models to examine myosin II roles and localization during mitochondrial fission. Fourth, how do other actin binding proteins contribute to mitochondrial dynamics? Evidence from other laboratories has suggested that cortactin, cofilin, and Arp2/3 complex play roles in assembling mitochondrially-associated actin, and that these factors might contribute to fission. I will use knock-out lines and dynamic localization by live-cell microscopy to test the relevance of these proteins in INF2-mediated actin assembly and mitochondrial fission.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professor Dr. Henry Higgs