FORMIN HOMOLOGY 2 DOMAIN BOUND TO THE BARBED END OF AN ACTIN FILAMENT

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Abstract: Formin proteins are potent regulators of actin dynamics. They are large, multidomain proteins that are implicated in a wide range of cellular processes such as cell polarization, adhesion and cytokinesis. These proteins are characterized by a conserved formin homology 2 (FH2) domain that mediates interactions with actin. The FH2 domain nucleates unbranched actin filaments and binds tightly to the filament barbed end. Kinetic studies of actin polymerization, as well as real-time imaging of FH2-bound filament growth, suggest that the FH2 domains remain stably bound to the actin filament as the actin monomers add onto or depolymerize from the barbed ends, leading to their description as leaky or processive caps. The crystal structure of an FH2 domain bound to actin monomers (determined in the Rosen lab) led to a model in which the FH2 domain exists in a rapidly equilibrating mixture of two different conformations at the barbed end of a filament. In this "nucleating ratchet" model, actin monomers add to one of these conformations (accessible) and dissociate from the other (blocked) (see fig5 in the relevant publications below). To understand the mechanism of processive capping by FH2 domain, I recently created FH2 mutants that stably bind the barbed end of filaments, but completely block barbed end elongation and depolymerization. We hypothesize that one class of these mutants is locked in the accessible conformation and a second class is locked in blocked conformation. The goals of my research are to determine three-dimensional images of the FH2 mutants bound to actin filaments using cryo-electron microscopy (cryo-EM) and image analysis. Ultimately, I would like to determine a three-dimensional image of the wild type FH2 domain bound to actin filaments and learn whether it can be described as a distribution of the conformations observed for the mutants. The actin cytoskeleton is a highly dynamic structure that is involved in a large number of cellular processes, ranging from maintenance of cell shape and polarity, to cell motility and cell division. Abnormalities in actin dynamics are associated with a variety of human diseases, such as cardiovascular diseases, neurodegeneration, and cancer (invasion and metastasis). Actin dynamics are regulated through the action of a number of interaction partners, many of which are conserved from yeast to humans. A better understanding of the function of these regulatory proteins will expand our knowledge of the fundamental mechanisms of actin dynamics, and potentially, will provide benefits for diagnosis and therapy of human diseases.