Mechanistic principles of Rho GTPase patterning
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Date
2024
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Abstract
Rho GTPases are regulators of morphogenesis in all eukaryotic cells. These peripheral membrane proteins spatially control actin cytoskeleton dynamics by locally concentrating at the plasma membrane in polarized activity patterns. However, the biochemical mechanisms by which Rho GTPases enrich at specific membrane sites where they are subsequently activated remain unknown. Here, I use in vitro reconstitution in model membrane systems to evaluate the relationship between the membrane-cytosolic shuttling of Rho GTPases and their activity state. I established a minimal system of templated Rho GTPase activity patterns that fully recapitulates the catalytic and spatial cycles of Rho GTPases. By utilizing this system, I demonstrate that effector proteins selectively stabilize active Rho GTPases on membrane surfaces. This stabilization is achieved through an avidity-driven mechanism that increases membrane retention of active Rho GTPases and decreases their diffusivity on the membrane. Together, the results show that effector proteins couple the catalytic and spatial cycles of Rho GTPases, thereby actively contributing to their spatial polarization. These findings are vital in our reconstitution of a synthetic Rho GTPase signaling network capable of spontaneous and stable polarization. To this end, I biochemically engineered and purified synthetic proteins, mediating regulatory feedback between distinct Rho GTPases based on direct recruitment. This linear feedback can promote the autonomous formation of stable Rho GTPase activity patterns, as shown by theory. I established a pipeline of biochemical assays to characterize these synthetic proteins, demonstrating their capacity to interact with active Rho GTPases and modulate their activity. Hence, I successfully created synthetic regulators of Rho GTPase activity that are directly recruited by active Rho GTPases. This paves the way for the implementation of regulatory feedback in an experimental system and is the first step toward a complete reconstitution of a minimal system that promotes the stable polarization of Rho GTPase activity.
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GTPase, GDI, Effectors, Patterning, Self-organization, Reconstitution