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Liquid-liquid phase separation of proteins has recently been observed on the surfaces of biological membranes, where it plays a role in diverse cellular processes, from assembly of focal adhesions and the immunological synapse, to biogenesis of trafficking vesicles. Interestingly in each of these cases, proteins on both surfaces of the membrane are thought to participate, suggesting that protein phase separation could be coupled across the membrane. To explore this possibility, we used an array of freestanding planar lipid membranes to observe protein phase separation simultaneously on both surfaces of lipid bilayers. When proteins known to engage in phase separation bound to the surfaces of these membranes, two-dimensional, protein-rich phases rapidly emerged. These phases displayed the hallmarks of a liquid, coarsening over time by fusing and re-rounding. Interestingly, we observed that protein-rich domains on one side of the membrane colocalized with those on the other side, resulting in transbilayer coupling. How do liquid-like protein phases communicate across the lipid bilayer? Our results, based on lipid probe partitioning and the differential mobility of proteins and lipids, collectively suggest an entropic coupling mechanism, which relies on the ability of protein phase separation to locally reduce the entropy of the underlying lipid membrane, most likely by increasing lipid packing. Regions of reduced entropy then colocalize across the bilayer to minimize the overall free energy of the membrane. These findings suggest a previously unknown mechanism by which cellular signals originating from one side of the membrane, triggered by protein phase separation, can be transferred to the opposite side.

During clathrin-mediated endocytosis, dozens of proteins assemble into an interconnected network at the plasma membrane. As initiators of endocytosis, Eps15 and Fcho1/2 concentrate downstream components, while permitting dynamic rearrangement within the budding vesicle. How do initiator proteins meet these competing demands? Here we show that Eps15 and Fcho1/2 rely on weak, liquid-like interactions to catalyse endocytosis. In vitro, these weak interactions promote the assembly of protein droplets with liquid-like properties. To probe the physiological role of these liquid-like networks, we tuned the strength of initiator protein assembly in real time using light-inducible oligomerization of Eps15. Low light levels drove liquid-like assemblies, restoring normal rates of endocytosis in mammalian Eps15-knockout cells. By contrast, initiator proteins formed solid-like assemblies upon exposure to higher light levels, which stalled vesicle budding, probably owing to insufficient molecular rearrangement. These findings suggest that liquid-like assembly of initiator proteins provides an optimal catalytic platform for endocytosis. Phase separation promotes clathrin-mediated endocytosis; Day et al. show that Eps15 and Fcho1 rely on weak, liquid-like interactions to efficiently catalyse endocytosis.

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.SignificanceWith nearly half their surfaces covered by proteins, biological membranes are highly crowded. Rapid diffusion and collision of membrane-bound proteins generates substantial steric pressure that is capable of shaping membrane surfaces. Many proteins involved in membrane remodeling, are intrinsically disordered. Having large hydrodynamic radii, disordered domains could contribute substantially to membrane crowding. However, it is unclear to what extent they are capable of generating steric pressure, owing to their conformational flexibility. Toward resolving this uncertainty, we have measured steric pressure at membrane surfaces during dynamic membrane remodeling events. Our data indicate that disordered domains generate significant steric pressure through entropic and electrostatic mechanisms, suggesting that they may constitute a critical, yet previously neglected class of membrane remodeling proteins.

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.

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.

Most small molecule chemotherapeutics must cross one or more cellular membrane barriers to reach their biochemical targets. Owing to the relatively low solubility of chemotherapeutics in the lipid membrane environment, high doses are often required to achieve a therapeutic effect. The resulting systemic toxicity has motivated efforts to improve the efficiency of chemotherapeutic delivery to the cellular interior. Toward this end, liposomes containing lipids with cationic head groups have been shown to permeabilize cellular membranes, resulting in the more efficient release of encapsulated drugs into the cytoplasm. However, the high concentrations of cationic lipids required to achieve efficient delivery remain a key limitation, frequently resulting in toxicity. Toward overcoming this limitation, here, we investigate the ability of ternary lipid mixtures to enhance liposomal delivery. Specifically, we investigate the delivery of the chemotherapeutic, doxorubicin, using ternary liposomes that are homogeneous at physiological temperature but have the potential to undergo membrane phase separation upon contact with the cell surface. This approach, which relies upon the ability of membrane phase boundaries to promote drug release, provides a novel method for reducing the overall concentration of cationic lipids required for efficient delivery. Our results show that this approach improves the performance of doxorubicin by up to 5-fold in comparison to the delivery of the same drug by conventional liposomes. These data demonstrate that ternary lipid compositions and cationic lipids can be combined synergistically to substantially improve the efficiency of chemotherapeutic delivery in vitro.

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 dynamic behavior of monoclonal antibodies (mAbs) at high concentration provides insight into protein microstructure and protein-protein interactions (PPI) that influence solution viscosity and protein stability. At high concentration, interpretation of the collective-diffusion coefficient Dc, as determined by dynamic light scattering (DLS), is highly challenging given the complex hydrodynamics and PPI at close spacings. In contrast, self-diffusion of a tracer particle by Brownian motion is simpler to understand. Herein, we develop fluorescence correlation spectroscopy (FCS) for the measurement of the long-time self-diffusion of mAb2 over a wide range of concentrations and viscosities in multiple co-solute formulations with varying PPI. The normalized self-diffusion coefficient D0/Ds (equal to the microscopic relative viscosity ηeff/η0) was found to be smaller than η/η0. Smaller ratios of the microscopic to macroscopic viscosity (ηeff/η) are attributed to a combination of weaker PPI and less self-association. The interaction parameters extracted from fits of D0/Ds with a length scale dependent viscosity model agree with previous measurements of PPI by SLS and SAXS. Trends in the degree of self-association, estimated from ηeff/η with a microviscosity model, are consistent with oligomer sizes measured by SLS. Finally, measurements of collective diffusion and osmotic compressibility were combined with FCS data to demonstrate that the changes in self-diffusion between formulations are due primarily to changes in the protein-protein friction in these systems, and not to protein-solvent friction. Thus, FCS is a robust and accessible technique for measuring mAb self-diffusion, and, by extension, microviscosity, PPI and self-association that govern mAb solution dynamics.

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.

During clathrin-mediated endocytosis, dozens of proteins assemble into an interconnected network at the plasma membrane. As early initiators of endocytosis, Eps15 and Fcho1 are responsible for locally concentrating downstream components on the membrane surface. However, they must also permit dynamic rearrangement of proteins within the budding vesicle. How do initiator proteins meet these competing demands? Here we show that Eps15 and Fcho1 rely on weak, liquid-like interactions to efficiently catalyze endocytosis. In reconstitution experiments, these weak interactions promote the assembly of protein droplets with liquid-like properties, including rapid coalescence and dynamic exchange of protein components. To probe the physiological role of liquid-like interactions among initiator proteins, we tuned the strength of initiator protein assembly in real time using light-inducible oligomerization of Eps15. Low light levels drove initiator proteins into liquid-like assemblies, restoring normal rates of endocytosis in mammalian Eps15 knockout cells. In contrast, initiator proteins formed solid-like assemblies upon exposure to higher light levels. Assembly of these structures stalled vesicle budding, likely owing to insufficient molecular rearrangement. These findings suggest that liquid-like assembly of early initiator proteins provides an optimal catalytic platform for endocytosis.

During clathrin-mediated endocytosis, dozens of proteins assemble into an interconnected network at the plasma membrane. As initiators of endocytosis, Eps15 and Fcho1/2 concentrate downstream components, while permitting dynamic rearrangement within the budding vesicle. How do initiator proteins meet these competing demands? Here we show that Eps15 and Fcho1/2 rely on weak, liquid-like interactions to catalyse endocytosis. In vitro, these weak interactions promote the assembly of protein droplets with liquid-like properties. To probe the physiological role of these liquid-like networks, we tuned the strength of initiator protein assembly in real time using light-inducible oligomerization of Eps15. Low light levels drove liquid-like assemblies, restoring normal rates of endocytosis in mammalian Eps15-knockout cells. By contrast, initiator proteins formed solid-like assemblies upon exposure to higher light levels, which stalled vesicle budding, probably owing to insufficient molecular rearrangement. These findings suggest that liquid-like assembly of initiator proteins provides an optimal catalytic platform for endocytosis.

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.

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.

Endocytic uptake of receptors from the cell surface plays an important role in diverse processes from cell signaling to nutrient internalization. Understanding the mechanisms by which endocytic structures select receptors for internalization is of fundamental importance to our understanding of cellular physiology. Binding of receptors to the endocytic protein machinery is known to facilitate receptor loading into endocytic structures. However, many receptor species use the same small set of biochemical motifs to interact with the endocytic machinery, suggesting that receptors may compete for a limited number of binding sites within endocytic structures. Previous studies have shown that such competition can substantially modify receptor uptake. However, a predictive biophysical understanding of this phenomenon is currently lacking. Toward addressing this gap, here we employ quantitative imaging and statistical thermodynamics to measure and predict the competition between two distinct receptor species that are internalized simultaneously from the cell surface. Our studies demonstrate that when receptors compete for the same interactions with the endocytic machinery, their uptake is fundamentally coupled. Importantly, we find that these trends can be quantitatively predicted by a simple thermodynamic analysis. These results suggest that multiple receptor species reach an equilibrium partitioning between endocytic structures and the surrounding plasma membrane as the receptors compete for occupancy within dynamic endocytic structures. More broadly, this work provides a quantitative framework for predicting the impact of competition on receptor uptake, an effect which has the potential to physically couple signaling pathways that impact diverse aspects of cellular physiology.

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.

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 membrane-bound 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.

Conjugated polyelectrolytes (CPEs) are promising materials for generating optoelectronics devices under environmentally friendly processing conditions, but challenges remain to develop methods to define lateral features for improved junction interfaces and direct optoelectronic pathways. We describe here the potential to use a bottom-up approach that employs self-assembly in lipid membranes to form structures to template the selective adsorption of CPEs. Phase separation of gel phase anionic lipids and fluid phase phosphocholine lipids allowed the formation of negatively charged domain assemblies that selectively adsorb a cationic conjugated polyelectrolyte (P2). Spectroscopic studies found the adsorption of P2 to negatively charged membranes resulted in minimal structural change of the solution phase polymer but yielded an enhancement in fluorescence intensity (~50%) due to loss of quenching pathways. Fluorescence microscopy, dynamic light scattering, and AFM imaging were used to characterize the polymer-membrane interaction and the polymer-bound domain structures of the biphasic membranes. In addition to randomly formed circular gel phase domains, we also show that predefined features, such as straight lines, can be directed to form upon etched patterns on the substrate, thus providing potential routes toward the self-organization of optoelectronic architectures.

Photoelectron Photoion Coincidence (PEPICO) spectroscopy holds the promise of a universal, isomer-selective, and sensitive analytical technique for time-resolved quantitative analysis of bimolecular chemical reactions. Unfortunately, its low dynamic range of ∼103 has largely precluded its use for this purpose, where a dynamic range of at least 105 is generally required. This limitation is due to the false coincidence background common to all coincidence experiments, especially at high count rates. Electron/ion pairs emanating from separate ionization events but arriving within the ion time of flight (TOF) range of interest constitute the false coincidence background. Although this background has uniform intensity at every m/z value, the Poisson scatter in the false coincidence background obscures small signals. In this paper, temporal ion deflection coupled with a position-sensitive ion detector enables suppression of the false coincidence background, increasing the dynamic range in the PEPICO TOF mass spectrum by 2-3 orders of magnitude. The ions experience a time-dependent electric deflection field at a well-defined fraction of their time of flight. This deflection defines an m/z- and ionization-time dependent ion impact position for true coincidences, whereas false coincidences appear randomly outside this region and can be efficiently suppressed. When cold argon clusters are ionized, false coincidence suppression allows us to observe species up to Ar9+, whereas Ar4+ is the largest observable cluster under traditional operation. This advance provides mass-selected photoelectron spectra for fast, high sensitivity quantitative analysis of reacting systems.

Membrane deformation is necessary to generate endocytic vesicles, but the molecular mechanisms proposed to drive membrane bending are controversial. Stachowiak and Schmid et al. report that crowding of proteins at the membrane is sufficient to induce curvature in vitro.

We demonstrate the construction of novel protein-lipid assemblies through the design of a lipid-like molecule, DPIDA, endowed with tail-driven affinity for specific lipid membrane phases and head-driven affinity for specific proteins. In studies performed on giant unilamellar vesicles (GUVs) with varying mole fractions of dipalymitoylphosphatidylcholine (DPPC), cholesterol, and diphytanoylphosphatidyl choline (DPhPC), DPIDA selectively partitioned into the more ordered phases, either solid or liquid-ordered (L(o)) depending on membrane composition. Fluorescence imaging established the phase behavior of the resulting quaternary lipid system. Fluorescence correlation spectroscopy confirmed the fluidity of the L(o) phase containing DPIDA. In the presence of CuCl(2), the iminodiacetic acid (IDA) headgroup of DPIDA forms the Cu(II)-IDA complex that exhibits a high affinity for histidine residues. His-tagged proteins were bound specifically to domains enriched in DPIDA, demonstrating the capacity to target protein binding selectively to both solid and L(o) phases. Steric pressure from the crowding of surface-bound proteins transformed the domains into tubules with persistence lengths that depended on the phase state of the lipid domains.

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.

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.

A novel transformation of giant lipid vesicles to produce nanotubular structures was observed upon the binding of streptavidin to biotinylated membranes. Unlike membrane budding and tubulation processes caused by proteins involved with endocytosis and vesicle fusion, streptavidin is known to crystallize at near the isoelectric point (pI 5 to 6) into planar sheets against biotinylated films. We have found, however, that at neutral pH membranes of low bending rigidity (10kT), such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), spontaneously produce tubular structures with widths ranging from micrometers to below the diffraction limit (250 nm) and lengths spanning up to hundreds of micrometers. The nanotubes were typically held taut between surface-bound vesicles suggesting high membrane tension, yet the lipid nanotubes exhibited a fluidic nature that enabled the transport of entrained vesicles. Confocal microscopy confirmed the uniform coating of streptavidin over the vesicles and nanotubes. Giant vesicles composed of lipid membranes of higher bending energy exhibited only aggregation in the presence of streptavidin. Routes toward the development of these highly curved membrane structures are discussed in terms of general protein-membrane interactions.

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., Proteins that bend membranes often contain curvature-promoting structural motifs such as wedges or crescent-shaped domains. Busch et al. report that intrinsically disordered domains can also drive membrane curvature and provide evidence that steric pressure driven by protein crowding mediates this effect.

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.

Advances in technology for electrochemical energy storage require increased understanding of electrolyte/electrode interfaces, including the electric double layer structure, and processes involved in charging of the interface, and the incorporation of this understanding into quantitative models. Simplified models such as Helmholtz's electric double-layer (EDL) concept don't account for the molecular nature of ion distributions, solvents, and electrode surfaces and therefore cannot be used in predictive, high-fidelity simulations for device design. This report presents theoretical results from models that explicitly include the molecular nature of the electrical double layer and predict critical electrochemical quantities such as interfacial capacitance. It also describes development of experimental tools for probing molecular properties of electrochemical interfaces through optical spectroscopy. These optical experimental methods are designed to test our new theoretical models that provide descriptions of the electric double layer in unprecedented detail.

Femtosecond time-resolved photoelectron angular distributions (PADs) are measured for the first time in the molecular frame of a dissociating molecule. Various stages of the dissociation process, NO{sub 2}{yields} NO(C {pi}{sup 2})+O(P{sup 3}) , are probed using ionization of the NO(C {pi}{sup 2}) fragment to NO{sup +}( X {sigma}{sup +1}) . The PADs evolve from forward-backward asymmetric with respect to the dissociation axis at short time delays ({ =}1 ps). Changes in the PADs directly reflect the time-dependent separation and reorientation of the dissociating photofragments. (c)

We present the first results using a new technique that combines femtosecond pump–probe methods with energy- and angle-resolved photoelectron–photoion coincidence imaging. The dominant dissociative multiphoton ionization (DMI) pathway for NO2 at 375.3 nm is identified as three-photon excitation to a repulsive potential surface correlating to NO(C 2Π)+O(3P) followed by one-photon ionization to NO+(X 1Σ+). Dissociation along this surface is followed on a femtosecond timescale.

Pathogenic viruses are a primary threat to our national security and to the health and economy of our world. Effective defense strategies to combat viral infection and spread require the development of understanding of the mechanisms that these pathogens use to invade the host cell. We present in this report results of our research into viral particle recognition and fusion to cell membranes and the role that protein affinity and confinement in lipid domains plays in membrane curvature in cellular fusion and fission events. Herein, we describe 1) the assembly of the G attachment protein of Nipah virus using point mutation studies to define its role in viral particle fusion to the cell membrane, 2) how lateral pressure of membrane bound proteins induce curvature in model membrane systems, and 3) the role of membrane curvature in the selective partitioning of molecular receptors and specific affinity of associated proteins.

Biological imaging and assay technologies rely on fluorescent organic dyes as reporters for a number of interesting targets and processes. However, limitations of organic dyes such as small Stokes shifts, spectral overlap of emission signals with native biological fluorescence background, and photobleaching have all inhibited the development of highly sensitive assays. To overcome the limitations of organic dyes for bioassays, we propose to develop lanthanide-based luminescent dyes and demonstrate them for molecular reporting applications. This relatively new family of dyes was selected for their attractive spectral and chemical properties. Luminescence is imparted by the lanthanide atom and allows for relatively simple chemical structures that can be tailored to the application. The photophysical properties offer unique features such as narrow and non-overlapping emission bands, long luminescent lifetimes, and long wavelength emission, which enable significant sensitivity improvements over organic dyes through spectral and temporal gating of the luminescent signal.Growth in this field has been hindered due to the necessary advanced synthetic chemistry techniques and access to experts in biological assay development. Our strategy for the development of a new lanthanide-based fluorescent reporter system is based on chelation of the lanthanide metal center using absorbing chromophores. Our first strategy involves %22Click%22 chemistry tomore » develop 3-fold symmetric chelators and the other involves use of a new class of tetrapyrrole ligands called corroles. This two-pronged approach is geared towards the optimization of chromophores to enhance light output.« less

Ultrafast photodynamics in a 1,3,5‐hexatriene are studied using femtosecond time‐resolved photoionization and photoelectron spectroscopy. The trans and cis isomers have distinctly different dynamics following excitation at the S2 origin near 250 nm. An intermediate, presumably the S1 state, is observed for both trans and cis isomers with lifetimes of 270 fs and 730 fs, respectively. Time‐delayed photoelectron spectra of cis‐hexatriene determine a 300 fs time scale for vibrational energy redistribution within the intermediate S1 state.

Results are presented from femtosecond time‐resolved coherent Raman experiments in which we excite and monitor vibrational coherence in gas‐phase samples of benzene and 1,3,5‐hexatriene. Different physical mechanisms for coherence decay are seen in these two molecules. In benzene, where the Raman polarizability is largely isotropic, the Q branch of the vibrational Raman spectrum is the primary feature excited. Molecules in different rotational states have different Q‐branch transition frequencies due to vibration–rotation interaction. Thus, the macroscopic polarization that is observed in these experiments decays because it has many frequency components from molecules in different rotational states, and these frequency components go out of phase with each other. In 1,3,5‐hexatriene, the Raman excitation produces molecules in a coherent superposition of rotational states, through (O, P, R, and S branch) transitions that are strong due to the large anisotropy of the Raman polarizability. The coherent superposition of rotational states corresponds to initially spatially oriented, vibrationally excited, molecules that are freely rotating. The rotation of molecules away from the initial orientation is primarily responsible for the coherence decay in this case. These experiments produce large (∼10% efficiency) Raman shifted signals with modest excitation pulse energies (10 μJ) demonstrating the feasibility of this approach for a variety of gas phase studies.

We demonstrate the use of two-color resonant four-wave mixing (TC-RFWM) spectroscopy to detect gas-phase ammonia molecules. Appropriate choice of the two different excitation frequencies is shown to cause substantial enhancement in the third-order susceptibility, χ (3) , and a consequent increase both in spectral sensitivity and in selectivity as compared with the corresponding one-color degenerate four-wave mixing (DFWM) technique.

This paper presents results from femtosecond time-resolved studies of the ultrafast internal conversion in 1,3,5-hexatriene following excitation of the 1{sup 1}B{sub u} state at around 250 nm. The internal conversion process is investigated using time-delayed single and multiple photon ionization with 350 nm and 310 nm probe pulses. The initial step in the internal conversion is found to be very fast (

In this paper we describe a method, based on the laser‐induced grating technique, for studying the spectroscopy of vibrational overtone‐excited gas‐phase water. Two phase‐coherent visible laser beams whose frequencies are in the range of the third overtone of the OH stretch in water are crossed in the gas‐phase sample. As the wavelength of these excitation beams is scanned through individual rovibrational OH overtone transitions, vibrational energy is deposited into the water in a spatially sinusoidal pattern. A fixed‐frequency 266 nm probe laser beam is diffracted from the resultant transmission diffraction grating in water. We show that under collision‐free conditions, probe laser diffraction is observed from the initially excited grating, which is a necessary condition for using this technique to study the absorption spectroscopy of the vibrationally excited molecules. Under multiple collision conditions, a probe laser wavelength‐independent refractive index grating is formed within the bulk sample. In...

A mechanical view provides an attractive alternative for predicting the behavior of complex systems since it circumvents the resource-intensive requirements of atomistic models; however, it remains extremely challenging to characterize the mechanical responses of a system at the molecular level. Here, the structural distribution is proposed to be an effective means to extracting the molecular mechanical properties. End-to-end distance distributions for a series of short poly-L-proline peptides with the sequence P(n)CG(3)K-biotin (n = 8, 12, 15 and 24) were used to experimentally illustrate this new approach. High-resolution single-molecule Forster-type resonance energy transfer (FRET) experiments were carried out and the conformation-resolving power was characterized and discussed in the context of the conventional constant-time binning procedure for FRET data analysis. It was shown that the commonly adopted theoretical polymer models-including the worm-like chain, the freely jointed chain, and the self-avoiding chain-could not be distinguished by the averaged end-to-end distances, but could be ruled out using the molecular details gained by conformational distribution analysis because similar polymers of different sizes could respond to external forces differently. Specifically, by fitting the molecular conformational distribution to a semi-flexible polymer model, the effective persistence lengths for the series of short poly-L-proline peptides were found to be size-dependent with values of ~190 A, ~67 A, ~51 A, and ~76 A for n = 8, 12, 15, and 24, respectively. A comprehensive computational modeling was carried out to gain further insights for this surprising discovery. It was found that P(8) exists as the extended all-trans isomaer whereas P(12) and P(15) predominantly contained one proline residue in the cis conformation. P(24) exists as a mixture of one-cis (75%) and two-cis (25%) isomers where each isomer contributes to an experimentally resolvable conformational mode. This work demonstrates the resolving power of the distribution-based approach, and the capacity of integrating high-resolution single-molecule FRET experiments with molecular modeling to reveal detailed structural information about the conformation of molecules on the length scales relevant to the study of biological molecules.

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.

Spatially resolved measurements of electric fields at electrochemical interfaces would be a critical step toward further understanding and modeling the detailed structure of electric double layers. The goal of this project was to perform proof-of-principle experiments to demonstrate the use of field-sensitive dyes for optical measurements of fields in electrochemical systems. A confocal microscope was developed that provides sensitive detection of the lifetime and high resolution spectra of excited fluorescence for dyes tethered to electrically conductive surfaces. Excited state lifetimes for the dyes were measured and found to be relatively unquenched when linked to indium tin oxide, but strongly quenched on gold surfaces. However, our fluorescence detection is sufficiently sensitive to measure spectra of submonolayer dye coatings even when the fluorescence was strongly quenched. Further work to create dye labeled interfaces on flat, uniform and durable substrates is necessary to make electric field measurements at interfaces using field sensitive dyes.

The innate immune system represents our first line of defense against microbial pathogens, and in many cases is activated by recognition of pathogen cellular components (dsRNA, flagella, LPS, etc.) by cell surface membrane proteins known as toll-like receptors (TLRs). As the initial trigger for innate immune response activation, TLRs also represent a means by which we can effectively control or modulate inflammatory responses. This proposal focused on TLR4, which is the cell-surface receptor primarily responsible for initiating the innate immune response to lipopolysaccharide (LPS), a major component of the outer membrane envelope of gram-negative bacteria. The goal was to better understand TLR4 activation and associated membrane proximal events, in order to enhance the design of small molecule therapeutics to modulate immune activation. Our approach was to reconstitute the receptor in biomimetic systems in-vitro to allow study of the structure and dynamics with biophysical methods. Structural studies were initiated in the first year but were halted after the crystal structure of the dimerized receptor was published early in the second year of the program. Methods were developed to determine the association constant for oligomerization of the soluble receptor. LPS-induced oligomerization was observed to be a strong function of buffer conditions. In 20 mM Tris pH 8.0 with 200 mM NaCl, the onset of receptor oligomerization occurred at 0.2 uM TLR4/MD2 with E coli LPS Ra mutant in excess. However, in the presence of 0.5 uM CD14 and 0.5 uM LBP, the onset of receptor oligomerization was observed to be less than 10 nM TLR4/MD2. Several methods were pursued to study LPS-induced oligomerization of the membrane-bound receptor, including CryoEM, FRET, colocalization and codiffusion followed by TIRF, and fluorescence correlation spectroscopy. However, there approaches met with only limited success.

Understanding and defending against pathogenic viruses is an important public health and biodefense challenge. The focus of our LDRD project has been to uncover the mechanisms enveloped viruses use to identify and invade host cells. We have constructed interfaces between viral particles and synthetic lipid bilayers. This approach provides a minimal setting for investigating the initial events of host-virus interaction - (i) recognition of, and (ii) entry into the host via membrane fusion. This understanding could enable rational design of therapeutics that block viral entry as well as future construction of synthetic, non-proliferating sensors that detect live virus in the environment. We have observed fusion between synthetic lipid vesicles and Vesicular Stomatitis virus particles, and we have observed interactions between Nipah virus-like particles and supported lipid bilayers and giant unilamellar vesicles.

Cell membranes are dynamic substrates that achieve a diverse array of functions through multi-scale reconfigurations. We explore the morphological changes that occur upon protein interaction to model membrane systems that induce deformation of their planar structure to yield nanotube assemblies. In the two examples shown in this report we will describe the use of membrane adhesion and particle trajectory to form lipid nanotubes via mechanical stretching, and protein adsorption onto domains and the induction of membrane curvature through steric pressure. Through this work the relationship between membrane bending rigidity, protein affinity, and line tension of phase separated structures were examined and their relationship in biological membranes explored.

A new excited−state spectroscopic method is reported. It is a two−color laser−induced grating tecnique for detecting optical transitions of rovibronically excited molecules in the gas phase. (AIP)

We show that induced-grating/four-wave-mixing ultrashort-pulse autocorrelation techniques that use slowly responding media offer the same phase information as interferometric second-harmonic generation. We also show that autocorrelation traces from nearly all possible induced-grating/four-wave-mixing beam geometries provide this information, with all yielding the same theoretical result, an integral of a fourth-order electric-field coherence function. Such traces clearly reveal chirp and self-phase-modulation effects without high-frequency fringes. Experiments using a two-beam-coupling arrangement in photorefractive media illustrate these effects.