Protein interactions underlying actin-based motility

描述（由申请人提供）：了解树突成核和细胞运动背后的分子相互作用是理解细胞内细胞骨架功能的关键一步。该项目的目标是识别和表征肌动蛋白和肌动蛋白相关蛋白之间的分子相互作用，这些相互作用控制着肌动蛋白细胞骨架的形成、组织和动力学。这是一个复杂的系统，因此，重点将主要放在三种特定的蛋白质及其在树突成核过程中的作用上。

DESCRIPTION (provided by applicant): Understanding the molecular interactions underlying dendritic nucleation and cell motility is a key step in understanding the function of the cytoskeleton within the cell. The goal of this project is to identify and characterize the molecular interactions between actin and actin associated proteins that control the formation, organization and dynamics of the actin cytoskeleton. This is a complicated system, and for that reason, the emphasis will primarily be on three specific proteins and their role in the process of dendritic nucleation. The specific aims will be to elucidate the molecular details of (1) the kinetics of profilin binding to ADF/cofilin-bound monomers to promote nucleotide exchange, and the association of profilin bound actin to the barbed end of actin filaments, (2) the connection between actin filament conformation, the binding kinetics of ADF/cofilin, and the role of ADF/cofilin in filament severing and disassembly, and (3) the binding of Arp 2/3 complex to actin filaments, as well as the kinetics and thermodynamics of Arp 2/3 mediated nucleation, and the capping of pointed ends by the complex. These aims will be achieved through the combined use of experimental biochemistry and cryo-electron microscopy techniques with well-established computational methods and models. The biochemistry work will aim to detail the kinetics of these protein interactions as well as other properties of the system such as the average length of actin branches or numbers of actin filaments. The structure of the Arp 2/3 branched filament networks and filaments bound with ADF/cofilin will be detailed using cryo-electron microscopy. Through the use of time resolved imaging, we will be able to capture all stages of the binding, branching and severing events, and use these EM reconstructions to produce atomic-level models for how these proteins interact. The computational work is central to this proposal, and its role will be to connect these two sets of experimental data to gain an understanding of how the structure relates to the function of these proteins. Atomically-detailed protein structures and complexes, primarily resulting from our electron microscopy reconstructions, will be used in Brownian dynamics simulations to model the interactions and association kinetics of two or more molecules. These results will then be directly compared with the results from the biochemical experiments. In parallel sets of calculations, we will calculate binding affinities between the various proteins are relate these results to the structural changes observed in the time-resolved EM work. This work will give perspective, not only into dendritic nucleation, but the motility of bacteria such as Listeria, and the function of the cell as a whole.