My laboratory studies assembly and dynamics of actin-based structures in mammalian cells. Mammals possess at least 14 distinct actin-based structures, which assemble from a common pool of cytoplasmic actin monomers. Hence, rigorous mechanisms must exist to control the time and place of assembly for each structure. For many actin-based structures, assembly mechanisms are poorly understood. Formin proteins are actin assembly factors, and the 15 mammalian formin isoforms provide clear potential to regulate actin assembly in many cellular contexts. In the preceding grant period, my laboratory elucidated many of the biochemical properties of mammalian formins. Using a comparative approach, we found that the FH2 domains of all mammalian formins studied had three core activities on actin: 1) nucleation acceleration;2) elongation rate regulation;and 3) capping protein inhibition. In addition, we found that some FH2 domains bundled filaments. The bundling formin, FRL2, is a potent inducer of filopodia in cells, suggesting a cellular role for bundling activity. We also find that another formin, INF2, has the remarkable ability to accelerate both actin polymerization and depolymerization. In cells, INF2 is tightly bound to the endoplasmic reticulum (ER), mediated in part by C-terminal farnesylation. Disruption of INF2's depolymerization activity causes ER collapse and prolific actin filament accumulation around the collapsed ER. INF2 also binds tightly to microtubules (MTs), possibly providing a link between MTs and actin in some cellular contexts. In this proposal, I will use a combined biochemical/cellular approach to address assembly of formin-mediated cellular structures, using INF2 and FRL2 as models.
In Aim 1, I identify regions of INF2 and FRL2 responsible for specific biochemical activities: actin depolymerization, actin bundling, MT binding, and membrane binding.
In Aim 2, I elucidate regulatory mechanisms for these biochemical activities.
In Aim 3, I use RNAi techniques, along with mutants identified in Aim 1, to address the cellular mechanisms behind INF2 and FRL2 function.
These aims are vital to my long-term goal of understanding the cellular functions of these formins at a mechanistic level. I believe that this level of understanding will be reached when we can reconstitute the process in a cell-free system. This proposal initiates the reconstitution process.

Public Health Relevance

Actin filaments are vital components of mammalian cells, playing roles in almost all phases of physiology. This research investigates fundamental molecular mechanisms by which mammalian cells control assembly of actin filaments. Our findings provide new and exciting opportunities for therapies against pathologies involving malfunction of these mechanisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM069818-09
Application #
8291043
Study Section
Cell Structure and Function (CSF)
Program Officer
Deatherage, James F
Project Start
2004-02-01
Project End
2013-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
9
Fiscal Year
2012
Total Cost
$319,476
Indirect Cost
$117,276
Name
Dartmouth College
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755
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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