Polymerization of the protein actin into helical filaments powers the directed motility of eukaryotic cells and some pathogenic bacteria. Actin assembly also plays critical roles in endocytosis, cytokinesis, and establishment of cell polarity. The essential regulatory protein, cofilin, is one of four actin-binding proteins that precisely choreograph actin assembly and organization in living systems. It acts by severing filaments, which increases the concentration of filament ends available for subunit addition and dissociation, thereby accelerating overall actin network dynamics and reorganization. It is therefore of general medical importance to understand how cofilin fragments actin filaments. Although the effects of cofilin binding to actin filaments have been extensively studied, the molecular mechanism of how cofilin severs filaments, which have stiffness comparable to commercial laboratory plastics, remains a central and unresolved mystery of cellular actin cytoskeleton reorganization. Elucidating the cofilin severing mechanism demands a multi-disciplinary approach integrating biology, chemistry, physics and mathematical modeling. Proposed research efforts focus on identifying how specific cation binding, post-translational modification, competition with other regulatory proteins, and filament shape deformations modulate actin filament structure and severing by vertebrate cofilin. Five general hypotheses will be tested. The first is that vertebrate cofilin severs filaments by dissociating a specific filamen-associated cation that controls filament structure and mechanical properties. The second is that competitive displacement of cofilin by other filament binding proteins can promote cofilactin filament severing by introducing boundaries of bare and cofilin-decorated segments. The third is that phosphorylation enhances cooperative cofilin binding and inhibits severing, not by lowering cofilin occupancy along filaments, but by reducing the density of boundaries where severing can occur. The fourth is that contractile protein- driven deformations in filament shape enhance severing by cofilin. The fifth is that actin filaments can act as tension sensors that recruit or exclude cofilin depending on the magnitude and mode of filament shape deformation. We will integrate biochemical and biophysical approaches, including experimental manipulation of single filaments, with mathematical modeling and simulations to develop predictive molecular models of actin filament elasticity and fragmentation, and directly test hypotheses formulated from biochemical and biophysical analysis of cofilin-actin interactions completed during the prior award period. The proposed research activities will advance knowledge of actin filament physiology by providing multi-scale relationships between filament mechanics, structure, and the biological function (e.g. severing activity) of essential regulatory proteins. New experimental and methods of analysis readily applicable to other filament binding proteins will be developed. Novel insight regarding the relationship between actin filament elasticity, conformation and regulatory protein occupancy will emerge from the work.

Public Health Relevance

Polymerization of the protein actin into helical filaments powers the directed motility of eukaryotic cells and some pathogenic bacteria. The essential filament severing protein, cofilin, is one of four actin regulatory proteins that precisely choreograph actin assembly and organization in living systems. The proposed studies will establish fundamental relationships between the physical properties of actin filaments and cofilin function.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Gindhart, Joseph G
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Yale University
Schools of Medicine
New Haven
United States
Zip Code
Karlberg, Tobias; Hornyak, Peter; Pinto, Ana Filipa et al. (2018) 14-3-3 proteins activate Pseudomonas exotoxins-S and -T by chaperoning a hydrophobic surface. Nat Commun 9:3785
Katkar, Harshwardhan H; Davtyan, Aram; Durumeric, Aleksander E P et al. (2018) Insights into the Cooperative Nature of ATP Hydrolysis in Actin Filaments. Biophys J 115:1589-1602
Huehn, Andrew; Cao, Wenxiang; Elam, W Austin et al. (2018) The actin filament twist changes abruptly at boundaries between bare and cofilin-decorated segments. J Biol Chem 293:5377-5383
Schramm, Anthony C; Hocky, Glen M; Voth, Gregory A et al. (2017) Actin Filament Strain Promotes Severing and Cofilin Dissociation. Biophys J 112:2624-2633
Elam, W Austin; Cao, Wenxiang; Kang, Hyeran et al. (2017) Phosphomimetic S3D cofilin binds but only weakly severs actin filaments. J Biol Chem 292:19565-19579
Zimmermann, Dennis; Homa, Kaitlin E; Hocky, Glen M et al. (2017) Mechanoregulated inhibition of formin facilitates contractile actomyosin ring assembly. Nat Commun 8:703
Hocky, Glen M; Baker, Joseph L; Bradley, Michael J et al. (2016) Cations Stiffen Actin Filaments by Adhering a Key Structural Element to Adjacent Subunits. J Phys Chem B 120:4558-67
Ennomani, Hajer; Letort, Gaëlle; Guérin, Christophe et al. (2016) Architecture and Connectivity Govern Actin Network Contractility. Curr Biol 26:616-26
Wang, Baisheng; Boeckel, Göran R; Huynh, Larry et al. (2016) Neuronal Calcium Sensor 1 Has Two Variants with Distinct Calcium Binding Characteristics. PLoS One 11:e0161414
De La Cruz, Enrique M; Gardel, Margaret L (2015) Actin Mechanics and Fragmentation. J Biol Chem 290:17137-44

Showing the most recent 10 out of 31 publications