bottom-up-vitro-methods-to-assay-ultrastructural-organization

Bottom-Up <em>In Vitro</em> Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins

Membrane remodeling occurs constantly at the plasma membrane and within cellular organelles. To fully dissect the role of the environment (ionic ...

Sorbonne Université

Czech Academy of Sciences, BIOCEV

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). Biomimetic in vitro assays using giant unilamellar vesicles (GUVs) are relevant tools to dissect and reveal insights in proteins-lipids interactions, membrane mechanics and curvature sensitivity. GUVs doped with PI(4,5)P2 are challenging to prepare. This report is dedicated to optimize 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 Cδ1). Septins have thus been chosen as reporters to probe the proper incorporation of PI(4,5)P2 into giant vesicles. We have shown that electro-formation on platinum wires is the most appropriate method to achieve an optimal septin-lipid interaction resulting from an optimal PI(4,5)P2 incorporation for which, we have optimized the growth conditions. Finally, 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.

Septin-based readout of PI(4,5)P2 incorporation into membranes of giant unilamellar vesicles.

Septins are cytoskeletal filaments that assemble at the inner face of the plasma membrane. They are localized at constriction sites and impact membrane remodeling. We report in vitro tools to examine how yeast septins behave on curved and deformable membranes. Septins reshape the membranes of Giant Unilamellar Vesicles with the formation of periodic spikes, while flattening smaller vesicles. We show that membrane deformations are associated to preferential arrangement of septin filaments on specific curvatures. 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 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.

Alexandre Beber

Laboratoire Physico Chimie Curie,
Institut Curie,
PSL Research University,
Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University

Sorbonne Université

Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins

Alexandre Beber

Laboratoire Physico Chimie Curie,Institut Curie,PSL Research University,Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University

Sorbonne Université

Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins

Laboratoire Physico Chimie Curie,
Institut Curie,
PSL Research University,
Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University