Membrane fission by protein crowding Snead, Wilton T.; Hayden, Carl C.; Gadok, Avinash K. ...
Proceedings of the National Academy of Sciences,
04/2017, Letnik:
114, Številka:
16
Journal Article
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Membrane fission, which facilitates compartmentalization of biological processes into discrete, membrane-bound volumes, is essential for cellular life. Proteins with specific structural features ...including constricting rings, helical scaffolds, and hydrophobic membrane insertions are thought to be the primary drivers of fission. In contrast, here we report a mechanism of fission that is independent of protein structure—steric pressure among membranebound proteins. In particular, random collisions among crowded proteins generate substantial pressure, which if unbalanced on the opposite membrane surface can dramatically increase membrane curvature, leading to fission. Using the endocytic protein epsin1 N-terminal homology domain (ENTH), previously thought to drive fission by hydrophobic insertion, our results show that membrane coverage correlates equally with fission regardless of the hydrophobicity of insertions. Specifically, combining FRET-based measurements of membrane coverage with multiple, independent measurements of membrane vesiculation revealed that fission became spontaneous as steric pressure increased. Further, fission efficiency remained equally potent when helices were replaced by synthetic membrane-binding motifs. These data challenge the view that hydrophobic insertions drive membrane fission, suggesting instead that the role of insertions is to anchor proteins strongly to membrane surfaces, amplifying steric pressure. In line with these conclusions, even green fluorescent protein (GFP) was able to drive fission efficiently when bound to the membrane at high coverage. Our conclusions are further strengthened by the finding that intrinsically disordered proteins, which have large hydrodynamic radii yet lack a defined structure, drove fission with substantially greater potency than smaller, structured proteins.
Assembly of highly curved membrane structures is essential to cellular physiology. The prevailing view has been that proteins with curvature-promoting structural motifs, such as wedge-like ...amphipathic helices and crescent-shaped BAR domains, are required for bending membranes. Here we report that intrinsically disordered domains of the endocytic adaptor proteins, Epsin1 and AP180 are highly potent drivers of membrane curvature. This result is unexpected since intrinsically disordered domains lack a well-defined three-dimensional structure. However, in vitro measurements of membrane curvature and protein diffusivity demonstrate that the large hydrodynamic radii of these domains generate steric pressure that drives membrane bending. When disordered adaptor domains are expressed as transmembrane cargo in mammalian cells, they are excluded from clathrin-coated pits. We propose that a balance of steric pressure on the two surfaces of the membrane drives this exclusion. These results provide quantitative evidence for the influence of steric pressure on the content and assembly of curved cellular membrane structures.
Abstract
Liquid-liquid phase separation of proteins occurs on both surfaces of cellular membranes during diverse physiological processes. In vitro reconstitution could provide insight into the ...mechanisms underlying these events. However, most existing reconstitution techniques provide access to only one membrane surface, making it difficult to probe transmembrane phenomena. To study protein phase separation simultaneously on both membrane surfaces, we developed an array of freestanding planar lipid membranes. Interestingly, we observed that liquid-like protein condensates on one side of the membrane colocalized with those on the other side, resulting in transmembrane coupling. Our results, based on lipid probe partitioning and mobility of lipids, suggest that protein condensates locally reorganize membrane lipids, a process which could be explained by multiple effects. These findings suggest a mechanism by which signals originating on one side of a biological membrane, triggered by protein phase separation, can be transferred to the opposite side.
Deformation of lipid membranes into curved structures such as buds and tubules is essential to many cellular structures including endocytic pits and filopodia. Binding of specific proteins to lipid ...membranes has been shown to promote membrane bending during endocytosis and transport vesicle formation. Additionally, specific lipid species are found to colocalize with many curved membrane structures, inspiring ongoing exploration of a variety of roles for lipid domains in membrane bending. However, the specific mechanisms by which lipids and proteins collaborate to induce curvature remain unknown. Here we demonstrate a new mechanism for induction and amplification of lipid membrane curvature that relies on steric confinement of protein binding on membrane surfaces. Using giant lipid vesicles that contain domains with high affinity for his-tagged proteins, we show that protein crowding on lipid domain surfaces creates a protein layer that buckles outward, spontaneously bending the domain into stable buds and tubules. 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: domain size and membrane tension. Our results suggest the intriguing possibility that confining structures, such as lipid domains and protein lattices, can amplify membrane bending by concentrating the steric interactions between bound proteins. This observation highlights a fundamental physical mechanism for initiation and control of membrane bending that may help explain how lipids and proteins collaborate to create the highly curved structures observed in vivo.
WASP‐family proteins are known to promote assembly of branched actin networks by stimulating the filament‐nucleating activity of the Arp2/3 complex. Here, we show that WASP‐family proteins also ...function as polymerases that accelerate elongation of uncapped actin filaments. When clustered on a surface, WASP‐family proteins can drive branched actin networks to grow much faster than they could by direct incorporation of soluble monomers. This polymerase activity arises from the coordinated action of two regulatory sequences: (i) a WASP homology 2 (WH2) domain that binds actin, and (ii) a proline‐rich sequence that binds profilin–actin complexes. In the absence of profilin, WH2 domains are sufficient to accelerate filament elongation, but in the presence of profilin, proline‐rich sequences are required to support polymerase activity by (i) bringing polymerization‐competent actin monomers in proximity to growing filament ends, and (ii) promoting shuttling of actin monomers from profilin–actin complexes onto nearby WH2 domains. Unoccupied WH2 domains transiently associate with free filament ends, preventing their growth and dynamically tethering the branched actin network to the WASP‐family proteins that create it. Collaboration between WH2 and proline‐rich sequences thus strikes a balance between filament growth and tethering. Our work expands the number of critical roles that WASP‐family proteins play in the assembly of branched actin networks to at least three: (i) promoting dendritic nucleation; (ii) linking actin networks to membranes; and (iii) accelerating filament elongation.
Synopsis
WASP‐family proteins activate the Arp2/3 complex to create new actin filaments in branched networks. New data show that these proteins also accelerate elongation of nearby filaments by providing a source of polymerization‐competent actin.
Polymerase activity of WASP‐family proteins arises from the recruitment of actin monomers and profilin‐actin complexes to surfaces containing a high density of WASP‐family proteins.
Most actin subunits enter a branched filament network via the WASP‐family protein‐coated surface and not directly from solution.
Profilin controls whether an actin monomer interacts with the WH2 or the proline‐rich domain of WASP family proteins.
Coordination between Proline‐rich and WH2 sequences sets the balance between the network polymerase and tethering activity of WASP family proteins.
In addition to creating new actin filaments via the Arp2/3 complex, WASP‐family proteins promote actin filament growth by recruiting actin monomers and actin‐profilin complexes to membrane surfaces.
Curved membranes are an essential feature of dynamic cellular structures, including endocytic pits, filopodia protrusions and most organelles. It has been proposed that specialized proteins induce ...curvature by binding to membranes through two primary mechanisms: membrane scaffolding by curved proteins or complexes; and insertion of wedge-like amphipathic helices into the membrane. Recent computational studies have raised questions about the efficiency of the helix-insertion mechanism, predicting that proteins must cover nearly 100% of the membrane surface to generate high curvature, an improbable physiological situation. Thus, at present, we lack a sufficient physical explanation of how protein attachment bends membranes efficiently. On the basis of studies of epsin1 and AP180, proteins involved in clathrin-mediated endocytosis, we propose a third general mechanism for bending fluid cellular membranes: protein-protein crowding. By correlating membrane tubulation with measurements of protein densities on membrane surfaces, we demonstrate that lateral pressure generated by collisions between bound proteins drives bending. Whether proteins attach by inserting a helix or by binding lipid heads with an engineered tag, protein coverage above ~20% is sufficient to bend membranes. Consistent with this crowding mechanism, we find that even proteins unrelated to membrane curvature, such as green fluorescent protein (GFP), can bend membranes when sufficiently concentrated. These findings demonstrate a highly efficient mechanism by which the crowded protein environment on the surface of cellular membranes can contribute to membrane shape change.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Cellular membranes are densely covered by proteins. Steric pressure generated by protein collisions plays a significant role in shaping and curving biological membranes. However, no method currently ...exists for measuring steric pressure at membrane surfaces. Here, we developed a sensor based on Förster resonance energy transfer (FRET), which uses the principles of polymer physics to precisely detect changes in steric pressure. The sensor consists of a polyethylene glycol chain tethered to the membrane surface. The polymer has a donor fluorophore at its free end, such that FRET with acceptor fluorophores in the membrane provides a real-time readout of polymer extension. As a demonstration of the sensor, we measured the steric pressure generated by a model protein involved in membrane bending, the N-terminal homology domain (ENTH) of Epsin1. As the membrane becomes crowded by ENTH proteins, the polymer chain extends, increasing the fluorescence lifetime of the donor. Drawing on polymer theory, we use this change in lifetime to calculate steric pressure as a function of membrane coverage by ENTH, validating theoretical equations of state. Further, we find that ENTH's ability to break up larger vesicles into smaller ones correlates with steric pressure rather than the chemistry used to attach ENTH to the membrane surface. This result addresses a long-standing question about the molecular mechanisms of membrane remodeling. More broadly, this sensor makes it possible to measure steric pressure in situ during diverse biochemical events that occur on membrane surfaces, such as membrane remodeling, ligand-receptor binding, assembly of protein complexes, and changes in membrane organization.
Random orientation of molecules within a sample leads to blurred observations of chemical reactions studied from the laboratory perspective. Methods developed for the dynamic imaging of molecular ...structures and processes struggle with this, as measurements are optimally made in the molecular frame. We used laser alignment to transiently fix carbon disulfide molecules in space long enough to elucidate, in the molecular reference frame, details of ultrafast electronic-vibrational dynamics during a photochemical reaction. These three-dimensional photoelectron imaging results, combined with ongoing efforts in molecular alignment and orientation, presage a wide range of insights obtainable from time-resolved studies in the molecular frame.
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.
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