Nature of curvature coupling of amphiphysin with membranes depends on its bound density

Abstract

Cells are populated by a vast array of membrane-binding proteins that execute critical functions. Functions, like signaling and intracellular transport, require the abilities to bind to highly curved membranes and to trigger membrane deformation. Among these proteins is amphiphysin 1, implicated in clathrin-mediated endocytosis. It contains a Bin-Amphiphysin-Rvs membrane-binding domain with an N-terminal amphipathic helix that senses and generates membrane curvature. However, an understanding of the parameters distinguishing these two functions is missing. By pulling a highly curved nanotube of controlled radius from a giant vesicle in a solution containing amphiphysin, we observed that the action of the protein depends directly on its density on the membrane. At low densities of protein on the nearly flat vesicle, the distribution of proteins and the mechanical effects induced are described by a model based on spontaneous curvature induction. The tube radius and force are modified by protein binding but still depend on membrane tension. In the dilute limit, when practically no proteins were present on the vesicle, no mechanical effects were detected, but strong protein enrichment proportional to curvature was seen on the tube. At high densities, the radius is independent of tension and vesicle protein density, resulting from the formation of a scaffold around the tube. As a consequence, the scaling of the force with tension is modified. For the entire density range, protein was enriched on the tube as compared to the vesicle. Our approach shows that the strength of curvature sensing and mechanical effects on the tube depends on the protein density.curvature-inducing | curvature-sensing | membrane nanotube | membrane physics

Linear aggregation of proteins on the membrane as a prelude to membrane remodeling

“…S4), it appears that the optimal binding free energy of the N-BAR domain for the observation of linear aggregation is between −4.6 and −11.6 kcal/mol. These predictions are in agreement with binding affinity measurements for amphiphysin (∼−10 kcal/mol), an N-BAR protein known to induce budding and tubulation (27). Moreover, our results may explain why the reversal of charge in the amphipathic helix of a whole series of N-BAR proteins resulted in the absence of tubulation, despite binding to the membrane (38).…”

“…In aggregates, the curvature maximum is an order of magnitude higher, 0.103-0.163 nm −1 (r = 6-9 nm). This value is in striking agreement with values of intrinsic curvature induced by N-BAR proteins in the same density regime predicted with fluorescence microscopy (27). Our result demonstrates that local curvatures in aggregates are comparable with the size of the protein but are coupled to emerging buds of at least an order of magnitude larger length scales.…”

“…Moreover, unlike previous experimental and theoretical reports, in which the adhesion of spherical and cylindrical particles caused a concave membrane deformation and, therefore, negatively curved vesiculation similar in order of magnitude to the adhered particles (3,5,29,30), we observe budding on the same side of the protein. Our qualitative observations on the budding behavior, as well as our quantitative results (in terms of bud radii), are consistent with in vivo and in vitro experiments of curvatureinducing proteins (9,27,28,31,32).…”

“…Recently, by measuring the amount of N-BAR proteins on the surface of the membrane, quantitative microscopy demonstrated that the mechanical properties of the membrane show density-dependent behavior similar to that seen in our simulations (27). The results of these experiments also imply a cooperative action of proteins, indicating aggregation (43); however, such experiments could not resolve the dynamic behavior of proteins at the molecular level.…”

“…a single protein or dense N-BAR lattices were studied theoretically and experimentally (14,(23)(24)(25)(26), simulating the N-BAR protein behavior at low and intermediate surface densities provides microscopic insights into the transient structures during the membrane restructuring at physiologically relevant concentrations. This is especially important in light of recent experimental work revealing the difference in mechanics of membrane bending under low-and high-density regimes of N-BAR proteins (27) and showed that N-BAR's mode of action needs to be studied with precise control of the bound protein density. In this work, we demonstrate a self-assembly behavior of N-BAR proteins on the membrane and quantify the relationship between the protein concentration and the resulting membrane morphology.…”