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Cellular membranes, which are densely crowded by proteins, take on an elaborate array of highly curved shapes. Steric pressure generated by protein crowding plays a significant role in shaping membrane surfaces. It is increasingly clear that many proteins involved in membrane remodeling contain substantial regions of intrinsic disorder. These domains have large hydrodynamic radii, suggesting that they may contribute significantly to steric congestion on membrane surfaces. However, it has been unclear to what extent they are capable of generating steric pressure, owing to their conformational flexibility. To address this gap, we use a recently developed sensor based on Förster resonance energy transfer to measure steric pressure generated at membrane surfaces by the intrinsically disordered domain of the endocytic protein, AP180. We find that disordered domains generate substantial steric pressure that arises from both entropic and electrostatic components. Interestingly, this steric pressure is largely invariant with the molecular weight of the disordered domain, provided that coverage of the membrane surface is held constant. Moreover, equivalent levels of steric pressure result in equivalent degrees of membrane remodeling, regardless of protein molecular weight. This result, which is consistent with classical polymer scaling relationships for semi-dilute solutions, helps to explain the molecular and physical origins of steric pressure generation by intrinsically disordered domains. From a physiological perspective, these findings suggest that a broad range of membrane-associated disordered domains are likely to play a significant and previously unknown role in controlling membrane shape.

Recruitment of receptors into clathrin-coated structures is essential to signal transduction and nutrient uptake. Among the many receptors involved in these processes, a significant fraction forms dimers. Dimerization of identical partners has generally been thought to promote receptor recruitment for uptake because of increased affinity of the dimer for the endocytic machinery. But what happens when receptors with substantially different affinities for the endocytic machinery come together to form a heterodimer? Evidence from diverse receptor classes, including G-protein-coupled receptors and receptor tyrosine kinases, suggests that heterodimerization with a strongly recruited receptor can drive significant recruitment of a receptor that lacks direct interactions with the endocytic machinery. However, a systematic biophysical understanding of this effect has yet to be established. Motivated by the potential of such events to influence cell signaling, here, we investigate the impact of receptor heterodimerization on endocytic recruitment using a family of engineered model receptors. As expected, we find that dimerization of a weakly recruited receptor with a strongly recruited receptor promotes incorporation of the weakly recruited receptor to endocytic structures. However, the effectiveness of this collaborative mechanism depends heavily on the relative strengths of endocytic recruitment of the two receptors that make up the dimer. Specifically, as the strength of endocytic recruitment of the weakly recruited receptor approaches that of the strongly recruited receptor, monomers of each receptor compete with heterodimers for space within endocytic structures. In this regime, the presence of the strongly recruited receptor drives a reduction in incorporation of the weakly recruited receptor into clathrin-coated structures. Similarly, as the strength of the dimer bond between the two receptors is progressively weakened, competition begins to dominate over collaboration. Collectively, these results demonstrate that the impact of receptor heterodimerization on endocytic recruitment is controlled by a delicate balance between collaborative and competitive mechanisms.

The ability of proteins to assemble at sites of high membrane curvature is essential to diverse membrane remodeling processes, including clathrin-mediated endocytosis. Multiple adaptor proteins within the clathrin pathway have been shown to sense regions of high membrane curvature, leading to local recruitment of the clathrin coat. Because clathrin triskelia do not bind to the membrane directly, it has remained unclear whether the clathrin coat plays an active role in sensing membrane curvature or is passively recruited by adaptor proteins. Using a synthetic tag to assemble clathrin directly on membrane surfaces, here we show that clathrin is a strong sensor of membrane curvature, comparable with previously studied adaptor proteins. Interestingly, this sensitivity arises from clathrin assembly rather than from the properties of unassembled triskelia, suggesting that triskelia have preferred angles of interaction, as predicted by earlier structural data. Furthermore, when clathrin is recruited by adaptors, its curvature sensitivity is amplified by 2- to 10-fold, such that the resulting protein complex is up to 100 times more likely to assemble on a highly curved surface compared with a flatter one. This exquisite sensitivity points to a synergistic relationship between the coat and its adaptor proteins, which enables clathrin to pinpoint sites of high membrane curvature, an essential step in ensuring robust membrane traffic. More broadly, these findings suggest that protein networks, rather than individual protein domains, are likely the most potent drivers of membrane curvature sensing.

Receptor internalization by endocytosis regulates diverse cellular processes, from the rate of nutrient uptake to the timescale of essential signaling events. The established view is that internalization is tightly controlled by specific protein-binding interactions. However, recent work suggests that physical aspects of receptors influence the process in ways that cannot be explained by biochemistry alone. Specifically, work from several groups suggests that increasing the steric bulk of receptors may inhibit their uptake by multiple types of trafficking vesicles. How do biochemical and biophysical factors work together to control internalization? Here, we show that receptor uptake is well described by a thermodynamic trade-off between receptor-vesicle binding energy and the entropic cost of confining receptors within endocytic vesicles. Specifically, using large ligands to acutely increase the size of engineered variants of the transferrin receptor, we demonstrate that an increase in the steric bulk of a receptor dramatically decreases its probability of uptake by clathrin-coated structures. Further, in agreement with a simple thermodynamic analysis, all data collapse onto a single trend relating fractional occupancy of the endocytic structure to fractional occupancy of the surrounding plasma membrane, independent of receptor size. This fundamental scaling law provides a simple tool for predicting the impact of receptor expression level, steric bulk, and the size of endocytic structures on receptor uptake. More broadly, this work suggests that bulky ligands could be used to drive the accumulation of specific receptors at the plasma membrane surface, providing a biophysical tool for targeted modulation of signaling and metabolism from outside the cell.

The ability of proteins to sense membrane curvature is essential to diverse membrane remodeling processes including clathrin-mediated endocytosis. Multiple adaptor proteins within the clathrin pathway have been shown to assemble together at curved membrane sites, leading to local recruitment of the clathrin coat. Because clathrin does not bind to the membrane directly, it has remained unclear whether clathrin plays an active role in sensing curvature or is passively recruited by its adaptor proteins. Using a synthetic tag to assemble clathrin directly on membrane surfaces, here we show that clathrin is a strong sensor of membrane curvature, comparable to previously studied adaptor proteins. Interestingly, this sensitivity arises from clathrin assembly, rather than from the properties of unassembled triskelia, suggesting that triskelia have preferred angles of interaction, as predicted by earlier structural data. Further, when clathrin is recruited by adaptors, its curvature sensitivity is amplified by two to ten-fold, such that the resulting protein complex is up to 100 times more likely to assemble on a highly curved surface, compared to a flatter one. This exquisite sensitivity points to a synergistic relationship between the coat and its adaptor proteins, which enables clathrin to pinpoint sites of high membrane curvature, an essential step in ensuring robust membrane traffic. More broadly, these findings suggest that protein networks, rather than individual protein domains, are likely the critical drivers of membrane curvature sensing.

Receptor internalization by endocytosis controls diverse cellular processes from the rate of nutrient uptake to the timescale of essential signaling events. While many of the biochemical motifs that enable uptake have been thoroughly investigated, the physical factors that influence receptor internalization remain poorly understood. Specifically, recent work from several groups has demonstrated that the steric bulk of receptors inhibits their uptake by multiple types of trafficking vesicles including caveolae, COPII vesicles, and clathrin coated pits. However, the mechanism by which endocytic structures differentiate among receptors of different sizes to control their uptake remains unclear. Using bulky ligands to acutely increase receptor size, here we show that receptor uptake is governed by a thermodynamic balance between receptor-vesicle binding energy and the entropic cost of confining receptors within nascent vesicles. Specifically, we used quantitative fluorescence imaging to precisely map the distribution of receptors among clathrin-coated pits at the plasma membrane surface. In agreement with a simple thermodynamic model, these data show that the increased entropic cost of internalizing bulky ligands inhibits receptor uptake, driving accumulation of ligated receptors at the plasma membrane. Further, by examining the differential uptake of two receptors competing for space within the same population of endocytic structures, our results reveal the relative importance of receptor size and receptor affinity for endocytic sites. From a biological perspective, these results can predict how processes such as ligand binding and dimerization, which alter both the effective size of receptors and their biochemical affinity for endocytic structures, will impact receptor uptake rate. From an applied perspective, this work guides the design of bulky ligands that can drive the accumulation of specific receptors at the plasma membrane surface, a tool that may be useful for modulating diverse pathways in cell physiology and signaling.

Highly curved membranes are essential to many cellular functions, motivating an intense effort to discover the mechanisms that shape them. A conserved feature among many proteins that participate in membrane bending is an amphipathic alpha helix that inserts into one membrane leaflet, attaching the protein to the membrane. These insertions are thought to bend membranes by pushing lipid heads apart like a wedge. First reported for the Epsin 1 N-terminal Homology (ENTH) domain, a protein believed to drive curvature during clathrin-mediated endocytosis, this mechanism is thought to shape a wide range of membrane structures, from trafficking vesicles to viral envelopes. However, recent computational studies have questioned the efficiency of the insertion mechanism, predicting that proteins with amphipathic helices must cover nearly 100% of the membrane surface to generate high curvature, an improbable situation given that cellular membranes are densely populated with a diversity of proteins. How then do proteins with amphipathic helices drive efficient bending of cellular membranes? We show that Epsin1 bends membranes via protein-protein crowding rather than via helix insertion. By correlating membrane tubule formation with FRET (Forster Resonance Energy Transfer) lifetime-based measurements of ENTH density on membrane surfaces, we demonstrate that protein coverage above ∼20% is sufficient to bend membranes. Whether proteins attach by inserting a helix or by binding lipid heads with an engineered tag, lateral steric pressure generated by bound proteins drives bending. Our results suggest that Epsin1's helix insertion functions primarily to achieve high protein-membrane affinity, enabling binding in a crowded environment. These findings call for a reexamination of the insertion hypothesis and demonstrate a new and highly efficient alternative mechanism by which the crowded protein environment on the surface of cellular membranes can directly contribute to membrane shape change.

Deformation of lipid membranes into curved structures such as buds and tubules is essential to many cellular processes. Lipid micro-domains are thought to co-localize with many curved membrane structures, inspiring ongoing exploration of a variety of roles for domains in membrane bending.We examined the role of lipid domains in spatial confinement of protein binding and discovered a new mechanism for curvature amplification that relies on global coupling. We formed giant unilamellar vesicles that contained insoluble lipid domains that strongly bound his-tagged proteins. We show that protein crowding on domain surfaces creates a protein layer that buckles outward, spontaneously bending the domain into stable, well-defined tubules as more proteins bind. In contrast to previously described bending mechanisms relying on local steric interactions between proteins and lipids (i.e. helix insertion into membranes), this mechanism produces tubules whose dimensions are defined by global parameters: binding energy and domain size. Our results suggest the intriguing possibility that domains can amplify membrane bending and define protrusion length scales by concentraing the steric interactions between the lipid bilayer and proteins. This mechanism may help explain the high curvatures induced by membrane bending proteins.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Two major mechanisms of cellular membrane bending during processes such as clathrin-mediated endocytosis have been previously proposed: bending by curved protein scaffolds such as a clathrin coat, and bending by insertion of wedge-like amphipathic helices into the membrane by adaptor proteins such as epsin1. Recently we have reported a third general membrane bending mechanism; bending by protein-protein crowding, where pressure generated by densely bound proteins drives membrane bending(1). Several endocytic adaptor proteins consist of a folded N-terminal membrane binding domain, and an unfolded C-terminal domain that binds clathrin and other proteins. Due to their lack of structure, the unfolded protein domains have much larger hydrodynamic radii than folded protein domains, potentially increasing the effects of their crowding compared to proteins of equal molecular weight. We have investigated the capability of these unfolded portions of adaptor proteins to bend membranes by binding them to giant unilamellar vesicles. using a Foster resonance energy tranfer based assay of protein density, developed in our previous studies, we find that the unstructured epsin C-terminus can bend model membranes at substantially lower densities than the structured epsin N-terminal homology domain, which has traditionally been thought to drive bending. These findings suggest that concentrating unfolded domains of adaptor proteins at endocytic sites may have a previously unappreciated role in promoting membrane bending. We also find that the addition of clathrin can locally increase the concentration of epsin1 on the membrane surface. Our ongoing experiments are investigating how clathrin and adaptor proteins work together to curve membrane surfaces.(1) Stachowiak, J.C. et al., Nat. Cell Biol. 14, 944, (2012).