In Vitro Reconstitution of Transcellular Tunnels Closure

Abstract

Several bacteria, such as Staphylococcus Aureus, are able to cross the endothelial barrier by inducing transcellular tunnels, called Transendothelial Macroapertures (TEM), in endothelial cells. The closure of these TEMs is critical to prevent endothelial permeability and cell death. Several proteins have been identified to play a role in this process. In particular, the I-BAR domain proteins MIM and ABBA have been shown to accumulate at the edge of the aperture shortly after the opening event. They subsequently recruit actin, followed by actin-rich membrane wave extension over the aperture. Interestingly, the related protein IRSp53, that unlike MIM and ABBA proteins does not have amphipathic alpha-helices on its I-BAR domain, has not been found at the edge of the TEM.The details of this mechanism remain unknown. Our objective is to characterize the physics underlying the first step in TEM closure. Our hypothesis is that MIM and ABBA have the ability to recognize the newly negatively-curved membrane at the edge of the TEM through their I-BAR domain. We use a minimal system where the protein is encapsulated in a giant unilamellar vesicle and can interact with the negatively-curved inner surface of a membrane tube that has been pulled out of the vesicle. We study protein-membrane interactions through fluorescence (confocal microscopy) and force (optical tweezers) measurements. By combining the two types of measurement, we quantify the affinity of the proteins for curved interfaces, ranging in radii of curvature from 10 to 100 nm, as well as their potential mechanical effect on the membrane. Our results show an original behavior where ABBA and IRSp53 are maximally enriched in membrane tubes of specific radii of curvature.