Liquid-like condensates mediate competition between actin branching and bundling.

Cellular remodeling of actin networks underlies cell motility during key morphological events, from embryogenesis to metastasis. In these transformations, there is an inherent competition between actin branching and bundling, because steric clashes among branches create a mechanical barrier to bundling. Recently, liquid-like condensates consisting purely of proteins involved in either branching or bundling of the cytoskeleton have been found to catalyze their respective functions. Yet in the cell, proteins that drive branching and bundling are present simultaneously. In this complex environment, which factors determine whether a condensate drives filaments to branch or become bundled? To answer this question, we added the branched actin nucleator, Arp2/3, to condensates composed of VASP, an actin bundling protein. At low actin to VASP ratios, branching activity, mediated by Arp2/3, robustly inhibited VASP-mediated bundling of filaments, in agreement with agent-based simulations. In contrast, as the actin to VASP ratio increased, addition of Arp2/3 led to formation of aster-shaped structures, in which bundled filaments emerged from a branched actin core, analogous to filopodia emerging from a branched lamellipodial network. These results demonstrate that multi-component, liquid-like condensates can modulate the inherent competition between bundled and branched actin morphologies, leading to organized, higher-order structures, similar to those found in motile cells.

Steric pressure between glycosylated transmembrane proteins inhibits internalization by endocytosis.

Clathrin-mediated endocytosis is essential for the removal of transmembrane proteins from the plasma membrane in all eukaryotic cells. Many transmembrane proteins are glycosylated. These proteins collectively comprise the glycocalyx, a sugar-rich layer at the cell surface, which is responsible for intercellular adhesion and recognition. Previous work has suggested that glycosylation of transmembrane proteins reduces their removal from the plasma membrane by endocytosis. However, the mechanism responsible for this effect remains unknown. To study the impact of glycosylation on endocytosis, we replaced the ectodomain of the transferrin receptor, a well-studied transmembrane protein that undergoes clathrin-mediated endocytosis, with the ectodomain of MUC1, which is highly glycosylated. When we expressed this transmembrane fusion protein in mammalian epithelial cells, we found that its recruitment to endocytic structures was substantially reduced in comparison to a version of the protein that lacked the MUC1 ectodomain. This reduction could not be explained by a loss of mobility on the cell surface or changes in endocytic dynamics. Instead, we found that the bulky MUC1 ectodomain presented a steric barrier to endocytosis. Specifically, the peptide backbone of the ectodomain and its glycosylation each made steric contributions, which drove comparable reductions in endocytosis. These results suggest that glycosylation constitutes a biophysical signal for retention of transmembrane proteins at the plasma membrane. This mechanism could be modulated in multiple disease states that exploit the glycocalyx, from cancer to atherosclerosis.

Lipid droplets as substrates for protein phase separation.

Membrane-associated protein phase separation plays critical roles in cell biology, driving essential cellular phenomena from immune signaling to membrane traffic. Importantly, by reducing dimensionality from three to two dimensions, lipid bilayers can nucleate phase separation at far lower concentrations compared with those required for phase separation in solution. How might other intracellular lipid substrates, such as lipid droplets, contribute to nucleation of phase separation? Distinct from bilayer membranes, lipid droplets consist of a phospholipid monolayer surrounding a core of neutral lipids, and they are energy storage organelles that protect cells from lipotoxicity and oxidative stress. Here, we show that intrinsically disordered proteins can undergo phase separation on the surface of synthetic and cell-derived lipid droplets. Specifically, we find that the model disordered domains FUS LC and LAF-1 RGG separate into protein-rich and protein-depleted phases on the surfaces of lipid droplets. Owing to the hydrophobic nature of interactions between FUS LC proteins, increasing ionic strength drives an increase in its phase separation on droplet surfaces. The opposite is true for LAF-1 RGG, owing to the electrostatic nature of its interprotein interactions. In both cases, protein-rich phases on the surfaces of synthetic and cell-derived lipid droplets demonstrate molecular mobility indicative of a liquid-like state. Our results show that lipid droplets can nucleate protein condensates, suggesting that protein phase separation could be key in organizing biological processes involving lipid droplets.

Morphological control of bundled actin networks subject to fixed-mass depletion.

Depletion interactions are thought to significantly contribute to the organization of intracellular structures in the crowded cytosol. The strength of depletion interactions depends on physical parameters such as the depletant number density and the depletant size ratio. Cells are known to dynamically regulate these two parameters by varying the copy number of proteins of a wide distribution of sizes. However, mammalian cells are also known to keep the total protein mass density remarkably constant, to within 0.5% throughout the cell cycle. We thus ask how the strength of depletion interactions varies when the total depletant mass is held fixed, a.k.a. fixed-mass depletion. We answer this question via scaling arguments, as well as by studying depletion effects on networks of reconstituted semiflexible actin in silico and in vitro. We examine the maximum strength of the depletion interaction potential U∗ as a function of q, the size ratio between the depletant and the matter being depleted. We uncover a scaling relation U∗ ∼ qζ for two cases: fixed volume fraction φ and fixed mass density ρ. For fixed volume fraction, we report ζ < 0. For the fixed mass density case, we report ζ > 0, which suggests that the depletion interaction strength increases as the depletant size ratio is increased. To test this prediction, we prepared our filament networks at fixed mass concentrations with varying sizes of the depletant molecule poly(ethylene glycol) (PEG). We characterize the depletion interaction strength in our simulations via the mesh size. In experiments, we observe two distinct actin network morphologies, which we call weakly bundled and strongly bundled. We identify a mass concentration where different PEG depletant sizes lead to weakly bundled or strongly bundled morphologies. For these conditions, we find that the mesh size and intra-bundle spacing between filaments across the different morphologies do not show significant differences, while the dynamic light scattering relaxation time and storage modulus between the two states do show significant differences. Our results demonstrate the ability to tune actin network morphology and mechanics by controlling depletant size and give insights into depletion interaction mechanisms under the fixed-depletant-mass constraint relevant to living cells.

Membrane bending by protein phase separation.

Membrane bending is a ubiquitous cellular process that is required for membrane traffic, cell motility, organelle biogenesis, and cell division. Proteins that bind to membranes using specific structural features, such as wedge-like amphipathic helices and crescent-shaped scaffolds, are thought to be the primary drivers of membrane bending. However, many membrane-binding proteins have substantial regions of intrinsic disorder which lack a stable three-dimensional structure. Interestingly, many of these disordered domains have recently been found to form networks stabilized by weak, multivalent contacts, leading to assembly of protein liquid phases on membrane surfaces. Here we ask how membrane-associated protein liquids impact membrane curvature. We find that protein phase separation on the surfaces of synthetic and cell-derived membrane vesicles creates a substantial compressive stress in the plane of the membrane. This stress drives the membrane to bend inward, creating protein-lined membrane tubules. A simple mechanical model of this process accurately predicts the experimentally measured relationship between the rigidity of the membrane and the diameter of the membrane tubules. Discovery of this mechanism, which may be relevant to a broad range of cellular protrusions, illustrates that membrane remodeling is not exclusive to structured scaffolds but can also be driven by the rapidly emerging class of liquid-like protein networks that assemble at membranes.

Light-Activated Assembly of Connexon Nanopores in Synthetic Cells.

During developmental processes and wound healing, activation of living cells occurs with spatiotemporal precision and leads to rapid release of soluble molecular signals, allowing communication and coordination between neighbors. Nonliving systems capable of similar responsive release hold great promise for information transfer in materials and site-specific drug delivery. One nonliving system that offers a tunable platform for programming release is synthetic cells. Encased in a lipid bilayer structure, synthetic cells can be outfitted with molecular conduits that span the bilayer and lead to material exchange. While previous work expressing membrane pore proteins in synthetic cells demonstrated content exchange, user-defined control over release has remained elusive. In mammalian cells, connexon nanopore structures drive content release and have garnered significant interest since they can direct material exchange through intercellular contacts. Here, we focus on connexon nanopores and present activated release of material from synthetic cells in a light-sensitive fashion. To do this, we re-engineer connexon nanopores to assemble after post-translational processing by a protease. By encapsulating proteases in light-sensitive liposomes, we show that assembly of nanopores can be triggered by illumination, resulting in rapid release of molecules encapsulated within synthetic cells. Controlling connexon nanopore activity provides an opportunity for initiating communication with extracellular signals and for transferring molecular agents to the cytoplasm of living cells in a rapid, light-guided manner.

Molecular mechanisms of steric pressure generation and membrane remodeling by disordered 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.

Correction: A continuum membrane model can predict curvature sensing by helix insertion.

Correction for 'A continuum membrane model can predict curvature sensing by helix insertion' by Yiben Fu et al., Soft Matter, 2021, 17, 10649-10663, DOI: 10.1039/D1SM01333E.

Clathrin senses membrane curvature.

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.

Weakly Internalized Receptors Use Coated Vesicle Heterogeneity to Evade Competition during Endocytosis.

The uptake of receptors by clathrin-mediated endocytosis underlies signaling, nutrient import, and recycling of transmembrane proteins and lipids. In the complex, crowded environment of the plasma membrane, receptors are internalized when they bind to components of the clathrin coat, such as the major adaptor protein, AP2. Receptors with higher affinity for AP2 are known to be more strongly internalized compared to receptors with lower affinity. However, it remains unclear how receptors with different affinities compete for space within crowded endocytic structures. To address this question, we constructed receptors with varying affinities for AP2 and allowed them to compete against one another during internalization. As expected, the internalization of a receptor with high affinity for AP2 was reduced when it was coexpressed with a competing receptor of similar affinity. However, receptors of low affinity for AP2 were surprisingly difficult to displace from endocytic structures, even when expressed alongside receptors with much higher affinity. To understand how these low-affinity receptors are protected from competition, we looked at AP2 heterogeneity across clathrin-coated structures. When we examined structures with lower-than-average AP2 content, we found that they were relatively enriched in cargo of low affinity for AP2 and depleted of cargo with high affinity. These findings suggest that the heterogeneity of adaptor protein content across the population of endocytic structures enables the internalization of diverse receptors. Given the critical role that internalization plays in signaling, this effect may help to prevent strongly internalized receptors from interfering with the cell's ability to process signals from weakly internalized receptors.

A continuum membrane model can predict curvature sensing by helix insertion.

Protein domains, such as ENTH (epsin N-terminal homology) and BAR (bin/amphiphysin/rvs), contain amphipathic helices that drive preferential binding to curved membranes. However, predicting how the physical parameters of these domains control this 'curvature sensing' behavior is challenging due to the local membrane deformations generated by the nanoscopic helix on the surface of a large sphere. We here use a deformable continuum model that accounts for the physical properties of the membrane and the helix insertion to predict curvature sensing behavior, with direct validation against multiple experimental datasets. We show that the insertion can be modeled as a local change to the membrane's spontaneous curvature, cins0, producing excellent agreement with the energetics extracted from experiments on ENTH binding to vesicles and cylinders, and of ArfGAP helices to vesicles. For small vesicles with high curvature, the insertion lowers the membrane energy by relieving strain on a membrane that is far from its preferred curvature of zero. For larger vesicles, however, the insertion has the inverse effect, de-stabilizing the membrane by introducing more strain. We formulate here an empirical expression that accurately captures numerically calculated membrane energies as a function of both basic membrane properties (bending modulus κ and radius R) as well as stresses applied by the inserted helix (cins0 and area Ains). We therefore predict how these physical parameters will alter the energetics of helix binding to curved vesicles, which is an essential step in understanding their localization dynamics during membrane remodeling processes.

PD-1 siRNA-Encapsulated Solid Lipid Nanoparticles Downregulate PD-1 Expression by Macrophages and Inhibit Tumor Growth : PD-1 siRNA-Encapsulated Solid Lipid Nanoparticles.

The present study was designed to test the hypothesis that programmed cell death-1 (PD-1) siRNA can downregulate PD-1 expression in macrophages in culture and in tumor tissues in mice and inhibit tumor growth in a mouse model. PD-1 siRNA was encapsulated in solid lipid nanoparticles (SLNs), and the physical properties of the resultant SLNs were characterized. The ability of the PD-1 siRNA-SLNs to downregulate PD-1 expression was confirmed in J774A.1 macrophages in culture and in tumor tissues in mice. Moreover, the antitumor activity of the PD-1 siRNA-SLNs was evaluated in a mouse model. The PD-1 siRNA-SLNs were roughly spherical, and their particle size, polydispersity index, and zeta potential were 141 ± 5 nm, 0.17 ± 0.02, and 20.7 ± 4.7 mV, respectively, with an siRNA entrapment efficiency of 98.9%. The burst release of the PD-1 siRNA from the SLNs was minimal. The PD-1 siRNA-SLNs downregulated PD-1 expression on J774A.1 macrophage cell surface as well as in macrophages in B16-F10 tumors pre-established in mice. In mice with pre-established B16-F10 tumors, the PD-1 siRNA-SLNs significantly inhibited the tumor growth, as compared with siRNA-SLNs prepared with non-functional, negative control siRNA. In conclusion, the PD-1 siRNA-SLNs inhibited tumor growth, likely related to their ability to downregulate PD-1 expression by tumor-associated macrophage (TAMs).

A Förster Resonance Energy Transfer-Based Sensor of Steric Pressure on Membrane Surfaces.

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.

Membrane fission by protein crowding.

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.

Receptor Heterodimerization Modulates Endocytosis through Collaborative and Competitive Mechanisms.

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.

Molecular Mechanisms of Membrane Curvature Sensing by a Disordered Protein.

The ability of proteins to sense membrane curvature is essential for the initiation and assembly of curved membrane structures. Established mechanisms of curvature sensing rely on proteins with specific structural features. In contrast, it has recently been discovered that intrinsically disordered proteins, which lack a defined three-dimensional fold, can also be potent sensors of membrane curvature. How can an unstructured protein sense the structure of the membrane surface? Many disordered proteins that associate with membranes have two key physical features: a high degree of conformational entropy and a high net negative charge. Binding of such proteins to membrane surfaces results simultaneously in a decrease in conformational entropy and an increase in electrostatic repulsion by anionic lipids. Here we show that each of these effects gives rise to a distinct mechanism of curvature sensing. Specifically, as the curvature of the membrane increases, the steric hindrance between the disordered protein and membrane is reduced, leading to an increase in chain entropy. At the same time, increasing membrane curvature increases the average separation between anionic amino acids and lipids, creating an electrostatic preference for curved membranes. Using quantitative imaging of membrane vesicles, our results demonstrate that long disordered amino acid chains with low net charge sense curvature predominately through the entropic mechanism. In contrast, shorter, more highly charged amino acid chains rely largely on the electrostatic mechanism. These findings provide a roadmap for predicting and testing the curvature sensitivity of the large and diverse set of disordered proteins that function at cellular membranes.

Liposome-Mediated Chemotherapeutic Delivery Is Synergistically Enhanced by Ternary Lipid Compositions and Cationic Lipids.

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.

Molecular thermodynamics of receptor competition for endocytic uptake.

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.

Structured and intrinsically disordered domains within Amphiphysin1 work together to sense and drive membrane curvature.

Cellular membranes undergo remodeling during many cellular processes including endocytosis, cytoskeletal protrusion, and organelle biogenesis. During these events, specialized proteins sense and amplify fluctuations in membrane curvature to create stably curved architectures. Amphiphysin1 is a multi-domain protein containing an N-terminal crescent-shaped BAR (Bin/Amphiphysin/Rvs) domain and a C-terminal domain that is largely disordered. When studied in isolation, the BAR domain of Amphiphysin1 senses membrane curvature and generates membrane tubules. However, the disordered domain has been largely overlooked in these studies. Interestingly, our recent work has demonstrated that the disordered domain is capable of substantially amplifying the membrane remodeling ability of the BAR domain. However, the physical mechanisms responsible for these effects are presently unclear. Here we elucidated the functional role of the disordered domain by gradually truncating it, creating a family of mutant proteins, each of which contained the BAR domain and a fraction of the disordered domain. Using quantitative fluorescence and electron microscopy, our results indicate that the disordered domain contributes to membrane remodeling by making it more difficult for the protein to bind to and assemble on flat membrane surfaces. Specifically, we found that the disordered domain began to significantly impact membrane remodeling when its projected area exceeded that of the BAR domain. Once this threshold was crossed, steric interactions with the membrane surface and with neighboring disordered domains gave rise to increased curvature sensing and membrane vesiculation, respectively. These findings provide insight into the synergy between structured and disordered domains, each of which play important biophysical roles in membrane remodeling.

Self-diffusion of a highly concentrated monoclonal antibody by fluorescence correlation spectroscopy: insight into protein-protein interactions and self-association.

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.