Annexins A1 and A2 Accumulate and Are Immobilized at Cross-Linked Membrane-Membrane Interfaces.

Rapid membrane repair is required to ensure cell survival after rupture of the plasma membrane. The annexin family of proteins is involved in plasma membrane repair (PMR) and is activated by the influx of Ca2+ from the extracellular medium at the site of injury. Annexins A1 and A2 (ANXA1 and ANXA2, respectively) are structurally similar and bind to negatively charged phosphatidylserine (PS) to induce membrane cross-linking and to promote fusion, which are both essential processes that occur during membrane repair. The degree of annexin accumulation and the annexin mobility at cross-linked membranes are important aspects of ANXA1 and ANXA2 function in repair. Here, we quantify ANXA1- and ANXA2-induced membrane cross-linking between giant unilamellar vesicles (GUVs). Time-lapse measurements show that ANXA1 and ANXA2 can induce membrane cross-linking on a time scale compatible with PMR. Cross-linked membrane-membrane interfaces between the GUVs persist in time without fusion, and quantification of confocal microscopy images demonstrates that ANXA1, ANXA2, and, to a lesser extent, PS lipids accumulate at the double membrane interface. Fluorescence recovery after photobleaching shows that the annexins are fully immobilized at the double membrane interface, whereas PS lipids display a 75% decrease in mobility. In addition, the complete immobilization of annexins between two membranes indicates a high degree of network formation between annexins, suggesting that membrane cross-linking is mainly driven by protein-protein interactions.

Membrane rolling induced by bacterial toxins.

Membrane curvature effects are important in numerous cellular processes and many membrane interacting proteins induce spontaneous curvature upon membrane binding. Shiga and cholera toxins both belong to the AB5 family of toxins and consist of a toxic A subunit and a membrane-binding pentameric B subunit. Shiga and cholera toxins induce tubular membrane invaginations in cells and GUVs due to curvature effects and the toxins are known from MD simulations to induce curvature. Membrane invaginations have been linked to uptake of the toxins into cells. As a novel model system to experimentally characterize curvature-inducing proteins, we study the morphology induced in planar membrane patches. It was previously shown that annexins induce distinct morphologies in membrane patches including membrane rolling. In this study we show that the B subunits of Shiga and cholera toxins (STxB, CTxB) both induce roll-up of cell-sized membrane patches. Rolling starts from the free membrane edges of the patch and is completed within a few seconds. We characterize the branched roll morphology and find experimental estimates for the spontaneous curvature of the toxins based on the topography of rolls. The estimates are in agreement with previous MD simulations. We quantify the dynamics of rolling as induced by the toxins and demonstrate agreement with a theoretical model of the rolling dynamics. The model solves the equation of motion for a membrane roll and includes viscous drag and adhesion to the support. The results suggest that membrane rolling may be a general phenomenon displayed by many proteins that induce negative curvature in membranes with free edges.