Supported Tubulated Bilayers: A Novel System for Evaluating Protein-Mediated Membrane Remodeling

Abstract

Fusion and fission of cellular membranes involve dramatic, precisely regulated changes in membrane curvature mediated by a number of proteins whose mechanisms are not well understood. Despite several recent advances, current methods for investigating curvature sensing and generation in real time using well-controlled systems remain limited. We have developed a novel system based on supported lipid bilayers (SLBs) in which high ionic strength during lipid bilayer deposition results in incorporation of excess lipids in the bilayer, which results in the spontaneous formation of lipid tubules after sequentially washing with water and physiological ionic strength buffer solutions. We find that the process of tubule formation is the result of an ion-dependent spreading of the SLB; addition of a physiological ionic strength buffer solution free of divalent ions leads to expansion of the bilayer and formation of tubules, likely due to increased membrane tension. Conversely, the addition of divalent ions results in contraction of the membrane and a proportional loss of tubules. These ionic conditions can be tuned for each experiment, allowing investigators to control the extent of tubulation. We show the utility of these supported tubulated bilayers, which we term “STuBs,” with an investigation of Sar1, a small Ras family G-protein known to influence membrane curvature. The addition of Sar1 to tubulated bilayers results in both further tubulation and tubule fission, of which fission is shown to be more dominant based on quantification of wide-field fluorescence microscopy images. Individual tubule formation events are observed with polarized total internal reflection fluorescence microscopy (pTIRFM), an imaging method that allows for semi-quantitative measurements of membrane deformations. Overall, STuBs is a simple experimental system, useful for monitoring solute- and protein-mediated effects on membrane topology in aqueous media and in real-time, using widely available instrumentation.