Collaborative Research: Mechanics of Reconstituted Self-Organized Contractile Actomyosin Systems

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Many cellular functions rely on the cytoskeleton, a collection of dynamic networks of biopolymers that provide cells with their mechanical properties and abilities to change shape. These networks have diverse architectures, which often coexist in cells. Although studies of whole cells have enabled characterization of these networks, how they self-organize to give rise to their observed structures and dynamics remains an outstanding question. By reconstituting network structures and dynamics from purified components, this project seeks to define the essential elements for self-organization and enable well-controlled measurements and modeling to probe its mechanisms. To date, such reconstitution studies have largely focused on protein assemblies in bulk solution or on solid supports. However, cells are enclosed by a lipid bilayer membrane, which is thought to be essential for many cellular functions and for the mechanics underlying them. The membrane does not just confine molecular species; it also provides a boundary that can anchor cytoskeletal structures yet deform under typical forces in cells. The project will build on recent technological advances to study protein networks that self-organize into synthetic lipid vesicles. The Broader Impacts of the work include the intrinsic merit of research itself as all cells contain some form of cytoskeleton. Additional activities include training of high school students and their teachers, along with undergraduates and post-doctoral research fellows. The PIs will also contribute to an art installation on synthetic cells that is being developed at the Marine Biological Laboratory at Woods Hole. By systematically characterizing the structures and dynamics accessible to different compositions of cytoskeletal proteins within vesicles, the project will advance understanding of self-organization and force generation by actin networks and the roles membrane confinement and coupling play in these processes. The project will specifically focus on reconstituting features of cell division, in particular the formation and constriction of a contractile ring composed of filamentous actin and the motor protein myosin II, along with additional structural (e.g., anchoring and bundling) proteins. The foundation of our experimental strategy is a powerful platform for reconstituting cytoskeletal networks in giant unilamellar vesicles. This will be paired with coarse-grained simulations of cytoskeletal networks. The project will determine the essential elements for network/ring formation by characterizing the self-organization of mixtures of actin, actin-binding proteins (alpha-actinin, fascin, and/or fimbrin), and motor proteins (myosin and/or a truncated form of it) in the absence of specific membrane interactions. Then, strategies to assemble an actin ring in coexistence with an actin cortex at the membrane will be tested to investigate how membrane binding alters the architecture of actin networks. Finally, patterned motor activation will be used to drive membrane-associated network/ring contraction and vesicle constriction, and the resulting forces will be investigated. To achieve the greatest impact, research, education, and outreach objectives will be closely integrated. This project was co-funded by the Systems and Synthetic Biology, and the Cellular Dynamics and Functions programs, both in the Molecular and Cellular Biosciences Division.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.