Eukaryotic cells depend on actin fibrils to perform a large number of diverse functions, including cell division, adhesion, and movement. Their formation is carefully regulated by the cells, which employ a variety of mechanisms to control polymerization with spatial and temporal specificity. Such a system is governed by a complex network of protein interactions. The importance of this system in cellular function means it has a commensurately large importance in biological malfunction. Mutations in any component can result in genetic disease. Pathogens can also abuse these systems to aid in infection of healthy cells through several pathways. Cancer cell metastasis occurs when cells move themselves away from a tumorous body, and this results from up-regulation of the actin network machinery. A fuller understanding of actin assembly and disassembly is crucial for the treatment of human disease. One of the central components of this system is the actin-related protein complex Arp2/3, which consists of two proteins, Arp2 and Arp3, as well as 5 cofactor proteins ArpC1-5. Arp2/3 binds to a preexisting actin filament and forms the beginnings of a daughter filament, which branches off at a 70 angle. There is evidence that the Arp2/3 complex is by default in an inactive state, i.e.it does not initiate branching without interaction with other cellular components. Conversely, experiments suggest mechanisms that deactivate the complex, promoting debranching. Due to the size and complexity of this protein assembly, relatively few computational studies to date have investigated the Arp2/3 system. I propose to study three aspects of the activation/deactivation of Arp2/3 using computational methodologies to advance our understanding of recent experimental results on these proteins. First, I will study the effect of ion binding at the interface between Arp2/3 and actin. Recent wor suggests that there may be specific sites at the interface where Arp2/3 binds actin to begin a new filament where cations can bind. We will test the hypothesis that having ions in these sites stabilizes the interface and promotes polymerization. Second, I will study the way in which special cofactors called nucleation promoting factors (NPFs) work with Arp2/3 to promote branching by binding Arp2/3 and actin to bring them together and to cause a conformational change in Arp2/3 that allows it to form a good interface with new actin monomers. Third, I will study the effect on Arp2/3 conformation on its ability to transform ATP to ADP, which is one of the controls that promotes debranching. These studies will enhance our understanding at the atomic level of key mechanisms used by cells to control networks of actin fibrils. My work will produce methodological improvements, and my resulting data will aid in the interpretation of previous experiments as well as the design of new ones.
The Arp2/3 complex is a molecular assembly of proteins that builds up scaffolding for the cell by forming a branched network of another protein, actin. The Arp2/3 complex is abused by cancer cells to generate mobility, by bacterial cells to enter and move about in healthy cells, and genetic mutations in this system result in serious human maladies; hence a fuller understanding of the regulation of this system is important for the treatment of human disease. We propose a series of computational experiments in conjunction with work by expert biologists to improve our molecular level understanding of the way in which Arp2/3 is turned on and off.
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| 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 |
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| 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; Dannenhoffer-Lafage, Thomas; Voth, Gregory A (2017) Coarse-Grained Directed Simulation. J Chem Theory Comput 13:4593-4603 |
| Li, Yujie; Christensen, Jenna R; Homa, Kaitlin E et al. (2016) The F-actin bundler ?-actinin Ain1 is tailored for ring assembly and constriction during cytokinesis in fission yeast. Mol Biol Cell 27:1821-33 |
| 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 |
| Winkelman, Jonathan D; Suarez, Cristian; Hocky, Glen M et al. (2016) Fascin- and ?-Actinin-Bundled Networks Contain Intrinsic Structural Features that Drive Protein Sorting. Curr Biol 26:2697-2706 |
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