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In vitro study of membrane remodeling and curvature sensing at the micrometric scale by budding yeast septins

Abstract : Septins constitute a novel class of cytoskeletal proteins. Budding yeast septins self-assemble into non-polar filaments bound to the inner plasma membrane through specific interactions with L- α-phosphatidylinositol-4,5- bisphosphate (PI(4,5)P2). Septins localize at constriction sites during cytokinesis and impact membrane remodeling processes. We have analyzed a range of in vitro biomimetic tools to examine how yeast septins behave on curved and deformable membranes. In vitro assays using Giant Unilamellar Vesicles (GUVs) are relevant tools to reveal insights in proteins-lipids interactions, membrane mechanics and curvature sensitivity. GUVs doped with PI(4,5)P2 are challenging to prepare. We first optimized the incorporation of PI(4,5)P2 lipids into GUVs by probing the proteins-PI(4,5)P2 GUVs interactions. We show that the interaction between budding yeast septins and PI(4,5)P2 is more specific than using usual reporters (phospholipase C1). We have shown that electro-formation on platinum wires is the most appropriate method to achieve an optimal septin-lipid interaction. Besides, we have shown that PI(4,5)P2 GUVs have to be used within a few hours after their preparation. Indeed, over time, PI(4,5)P2 is expelled from the GUV membrane and the PI(4,5)P2 concentration in the bilayer decreases. Next, we analyzed how septins can control the mechanical properties of membranes and analyzed how membrane deformations could be induced by a specific curvature sensitivity of septins. Indeed, we have shown that septins reshape the membranes of Giant Unilamellar Vesicles with the formation of periodic spikes. We have shown that membrane deformations are associated to septin filament curvature arrangement preferences. When binding to bilayers supported on custom-designed periodic wavy patterns displaying positive and negative micrometric radii of curvatures, septin filaments remain straight and perpendicular to the curvature of the convex parts, while bending negatively to follow concave geometries. Based on these results, we propose a theoretical model that quantitatively describes the deformations and micrometric curvature sensitivity observed in vitro. The model captures the reorganizations of septin filaments throughout cytokinesis in vivo, providing mechanistic insights into cell division.
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Alexandre Beber. In vitro study of membrane remodeling and curvature sensing at the micrometric scale by budding yeast septins. Biological Physics [physics.bio-ph]. Sorbonne Université, 2018. English. ⟨NNT : 2018SORUS375⟩. ⟨tel-02015522v2⟩

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