While it is clear that cytoskeletal elements (actin and microtubules) play roles in the dynamics and integrity of intracellular organelles, the mechanisms by which these actions occur are uncertain. In the last grant period, my laboratory revealed exciting cellular functions for the mammalian formin protein INF2. Importantly, INF2 exists as two C-terminal splice variants that essentially act as distinct proteins. One splice variant, INF2-CAAX, is prenylated and tightly bound to endoplasmic reticulum (ER). We showed that INF2-CAAX functions in mitochondrial fission. INF2-CAAX polymerizes actin filaments at the ER/mitochondrial interface, and INF2-mediated actin polymerization stimulates recruitment of the dynamin-like GTPase Drp1 to mitochondria, leading to fission. A second INF2 splice variant, INF2- nonCAAX, is not prenylated and is found in an actin-dependent meshwork-like pattern in the cytoplasm. INF2-nonCAAX plays a role in generation of dynamic actin "patches" in the Golgi region, and our results suggest that these patches act in fission of Golgi and other organelles. Overall, our results suggest that INF2 is a novel actin-based membrane fission factor. In this grant period, we study how INF2's effects on actin translate into mitochondrial fission.
Aim 1 addresses the biochemical mechanism by which INF2 influences actin dynamics. INF2 is an unusual formin biochemically. While most formins accelerate actin polymerization, INF2 accelerates both polymerization and depolymerization. To depolymerize filaments, INF2 possesses a potent filament severing activity. In this aim, we elucidate INF2's severing and depolymerization mechanism using TIRF microscopy, Atomic Force Microscopy and biochemical techniques. We also use mutagenesis to determine important sequences for severing/depolymerization, and will use these mutants for experiments in Aims 2 and 3.
Aim 2 addresses the mechanism by which INF2 deforms membranes. Based on our preliminary results showing that mitochondrial fission requires myosin activity, we hypothesize that INF2-generated filaments are tracks for myosin-mediated contraction. In this aim, we use fixed-cell and live-cell microscopy to test this model.
Aim 3 addresses INF2 regulation. While other formins are regulated by auto-inhibition, our preliminary data suggest that an additional molecule is required for INF2 inhibition. Furthermore, we have evidence that Drp1 might be an inhibitory factor for INF2-CAAX. We use purified proteins and a novel cell-free assay system to elucidate Drp1's effect on INF2 and on actin, as well as to identify inhibitors for INF2-nonCAAX.

Public Health Relevance

The dynamic properties of mitochondria (fission, fusion, and movement) are currently major targets of disease research in neurodegeneration (Alzheimer's, Huntington's, Parkinson's, and other diseases). Recently, we found an important but hitherto unknown role for actin polymerization, through the protein INF2, in mitochondrial fission. This grant investigates the mechanistic basis for INF2's function in mitochondrial fission, as well as i the fission of other cellular membranes in which we suspect INF2 to be involved.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-CB-G (02))
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Gindhart, Joseph G
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Dartmouth College
Schools of Medicine
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Korobova, Farida; Gauvin, Timothy J; Higgs, Henry N (2014) A role for myosin II in mammalian mitochondrial fission. Curr Biol 24:409-14
Gurel, Pinar S; Hatch, Anna L; Higgs, Henry N (2014) Connecting the cytoskeleton to the endoplasmic reticulum and Golgi. Curr Biol 24:R660-72
Gurel, Pinar S; Ge, Peng; Grintsevich, Elena E et al. (2014) INF2-mediated severing through actin filament encirclement and disruption. Curr Biol 24:156-64
Sharma, Shivani; Grintsevich, Elena E; Woo, JungReem et al. (2014) Nanostructured self-assembly of inverted formin 2 (INF2) and F-actin-INF2 complexes revealed by atomic force microscopy. Langmuir 30:7533-9
Thompson, Morgan E; Heimsath, Ernest G; Gauvin, Timothy J et al. (2013) FMNL3 FH2-actin structure gives insight into formin-mediated actin nucleation and elongation. Nat Struct Mol Biol 20:111-8
Korobova, Farida; Ramabhadran, Vinay; Higgs, Henry N (2013) An actin-dependent step in mitochondrial fission mediated by the ER-associated formin INF2. Science 339:464-7
Ramabhadran, Vinay; Hatch, Anna L; Higgs, Henry N (2013) Actin monomers activate inverted formin 2 by competing with its autoinhibitory interaction. J Biol Chem 288:26847-55
Heimsath Jr, Ernest G; Higgs, Henry N (2012) The C terminus of formin FMNL3 accelerates actin polymerization and contains a WH2 domain-like sequence that binds both monomers and filament barbed ends. J Biol Chem 287:3087-98
Hetheridge, Clare; Scott, Alice N; Swain, Rajeeb K et al. (2012) The formin FMNL3 is a cytoskeletal regulator of angiogenesis. J Cell Sci 125:1420-8
Sun, Hua; Schlondorff, Johannes S; Brown, Elizabeth J et al. (2011) Rho activation of mDia formins is modulated by an interaction with inverted formin 2 (INF2). Proc Natl Acad Sci U S A 108:2933-8

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