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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Chakrabarti, Rajarshi; Ji, Wei-Ke; Stan, Radu V et al. (2018) INF2-mediated actin polymerization at the ER stimulates mitochondrial calcium uptake, inner membrane constriction, and division. J Cell Biol 217:251-268
Ji, Wei-Ke; Chakrabarti, Rajarshi; Fan, Xintao et al. (2017) Receptor-mediated Drp1 oligomerization on endoplasmic reticulum. J Cell Biol 216:4123-4139
Hatch, Anna L; Ji, Wei-Ke; Merrill, Ronald A et al. (2016) Actin filaments as dynamic reservoirs for Drp1 recruitment. Mol Biol Cell 27:3109-3121
Ji, Wei-ke; Hatch, Anna L; Merrill, Ronald A et al. (2015) Actin filaments target the oligomeric maturation of the dynamin GTPase Drp1 to mitochondrial fission sites. Elife 4:e11553
Gurel, Pinar S; A, Mu; Guo, Bingqian et al. (2015) Assembly and turnover of short actin filaments by the formin INF2 and profilin. J Biol Chem 290:22494-506
Gauvin, Timothy J; Young, Lorna E; Higgs, Henry N (2015) The formin FMNL3 assembles plasma membrane protrusions that participate in cell-cell adhesion. Mol Biol Cell 26:467-77
Young, Lorna E; Heimsath, Ernest G; Higgs, Henry N (2015) Cell type-dependent mechanisms for formin-mediated assembly of filopodia. Mol Biol Cell 26:4646-59
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-R672
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

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