Browsing by Subject "Liquid-liquid phase separation"

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Intrinsically disordered proteins as mechanistic drivers of membrane remodeling and endocytosis
(2023-12) Yuan, Feng, Ph. D.; Stachowiak, Jeanne Casstevens; Parekh, Sapun; Senning, Eric; Wang, Evan
Membrane bending is a ubiquitous cellular process that is required for membrane traffic, cell motility, organelle biogenesis, and cell division. Proteins that bind to membranes using specific structural features, such as wedge-like amphipathic helices and crescent-shaped scaffolds, are thought to be the primary drivers of membrane bending. However, many membrane-binding proteins have substantial regions of intrinsic disorder which lack a stable three-dimensional structure. Recently, our group and others have reported that intrinsically disordered proteins can also be potent drivers of membrane bending. Specifically, when noninteracting disordered domains are crowded together in cellular structures, steric repulsion among them drives the membrane to buckle outward, taking on a curved shape. Interestingly, rather than repelling one another, many of these disordered domains have recently been found to form networks stabilized by weak, multivalent contacts, leading to assembly of protein liquid phases on membrane surfaces. In my thesis, I first characterized the impact of protein liquid-liquid phase separation (LLPS) on membrane curvature. Specifically, I have demonstrated that protein phase separation on the surfaces of synthetic and cell-derived membrane vesicles creates a substantial compressive stress in the plane of the membrane. This stress drives the membrane to bend inward, creating protein-lined membrane tubules. Discovery of this mechanism, which may be relevant to a broad range of cellular protrusions, illustrates that membrane remodeling can also be driven by the rapidly emerging class of liquid-like protein networks that assemble at membranes. To further understand how repulsive and attractive domains work together to apply bending stresses to the membrane surface, I then investigated series of disordered protein chimeras, which combine protein domains previously shown to drive either convex or concave membrane curvature. Using these chimeras, I demonstrated that disordered protein layers with opposite curvature preferences can either work together to amplify curvature or can oppose one another to create context-dependent control of membrane shape. This work outlines a set of design rules that can be used to understand the impact of disordered proteins on membrane curvature. Furthermore, I studied how LLPS impacts endocytosis. I examined the influence of ubiquitin on the stability of the liquid endocytic protein network. In vitro, I found that recruitment of small amounts of polyubiquitin dramatically increased the stability of Eps15 condensates, suggesting that ubiquitylation could nucleate endocytic sites. In live cell imaging experiments, a version of Eps15 that lacked the ubiquitin-interacting motif failed to rescue defects in endocytic initiation created by Eps15 knockout. Furthermore, fusion of Eps15 to a deubiquitinase enzyme destabilized nascent endocytic sites within minutes. These results suggest that ubiquitylation drives assembly of the flexible protein network responsible for catalyzing endocytic events. Collectively, my thesis work has illustrated biophysical mechanisms by which intrinsically disordered proteins could regulate membrane bending and endocytosis.