Membrane surfaces regulate assembly of ribonucleoprotein condensates.

Biomolecular condensates organize biochemistry, yet little is known about how cells control the position and scale of these structures. In cells, condensates often appear as relatively small assemblies that do not coarsen into a single droplet despite their propensity to fuse. Here, we report that ribonucleoprotein condensates of the glutamine-rich protein Whi3 interact with the endoplasmic reticulum, which prompted us to examine how membrane association controls condensate size. Reconstitution revealed that membrane recruitment promotes Whi3 condensation under physiological conditions. These assemblies rapidly arrest, resembling size distributions seen in cells. The temporal ordering of molecular interactions and the slow diffusion of membrane-bound complexes can limit condensate size. Our experiments reveal a trade-off between locally enhanced protein concentration at membranes, which favours condensation, and an accompanying reduction in diffusion, which restricts coarsening. Given that many condensates bind endomembranes, we predict that the biophysical properties of lipid bilayers are key for controlling condensate sizes throughout the cell.

Dilute phase oligomerization can oppose phase separation and modulate material properties of a ribonucleoprotein condensate.

SignificanceA large subclass of biomolecular condensates are linked to RNA regulation and are known as ribonucleoprotein (RNP) bodies. While extensive work has identified driving forces for biomolecular condensate formation, relatively little is known about forces that oppose assembly. Here, using a fungal RNP protein, Whi3, we show that a portion of its intrinsically disordered, glutamine-rich region modulates phase separation by forming transient alpha helical structures that promote the assembly of dilute phase oligomers. These oligomers detour Whi3 proteins from condensates, thereby impacting the driving forces for phase separation, the protein-to-RNA ratio in condensates, and the material properties of condensates. Our findings show how nanoscale conformational and oligomerization equilibria can influence mesoscale phase equilibria.

Getting droplets into shape.

Protein clusters at interfaces control sizes and properties of biomolecular condensates.

The Control Centers of Biomolecular Phase Separation: How Membrane Surfaces, PTMs, and Active Processes Regulate Condensation.

Biomolecular condensation is emerging as an essential process for cellular compartmentalization. The formation of biomolecular condensates can be driven by liquid-liquid phase separation, which arises from weak, multivalent interactions among proteins and nucleic acids. A substantial body of recent work has revealed that diverse cellular processes rely on biomolecular condensation and that aberrant phase separation may cause disease. Many proteins display an intrinsic propensity to undergo phase separation. However, the mechanisms by which cells regulate phase separation to build functional condensates at the appropriate time and location are only beginning to be understood. Here, we review three key cellular mechanisms that enable the control of biomolecular phase separation: membrane surfaces, post-translational modifications, and active processes. We discuss how these mechanisms may function in concert to provide robust control over biomolecular condensates and suggest new research avenues that will elucidate how cells build and maintain these key centers of cellular compartmentalization.

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.

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.

BAR scaffolds drive membrane fission by crowding disordered domains.

Cellular membranes are continuously remodeled. The crescent-shaped bin-amphiphysin-rvs (BAR) domains remodel membranes in multiple cellular pathways. Based on studies of isolated BAR domains in vitro, the current paradigm is that BAR domain-containing proteins polymerize into cylindrical scaffolds that stabilize lipid tubules. But in nature, proteins that contain BAR domains often also contain large intrinsically disordered regions. Using in vitro and live cell assays, here we show that full-length BAR domain-containing proteins, rather than stabilizing membrane tubules, are instead surprisingly potent drivers of membrane fission. Specifically, when BAR scaffolds assemble at membrane surfaces, their bulky disordered domains become crowded, generating steric pressure that destabilizes lipid tubules. More broadly, we observe this behavior with BAR domains that have a range of curvatures. These data suggest that the ability to concentrate disordered domains is a key driver of membrane remodeling and fission by BAR domain-containing proteins.

A Tethered Vesicle Assay for High-Throughput Quantification of Membrane Fission.

Membrane fission, which divides membrane surfaces into separate compartments, is essential to diverse cellular processes including membrane trafficking and cell division. Quantitative assays are needed to elucidate the physical mechanisms by which proteins drive membrane fission. Toward this goal, several experimental tools have been developed, including visualizing fission products using electron microscopy, measuring membrane shedding from a lipid reservoir, and observing fission of individual membrane tubes pulled from giant vesicles. However, no existing assay of membrane fission provides a quantitative, high-throughput measure of the distribution of vesicle curvatures generated by fission-driving proteins. Toward addressing this challenge, here we describe a novel approach that uses confocal fluorescence imaging to quantify the diameter distribution of membrane vesicles that have been tethered to a coverslip surface following exposure to fission-driving proteins. We employ this assay to measure the progressive appearance of high curvature fission products upon exposure of vesicles to increasing protein concentration. Results from this approach are in quantitative agreement with measurements from electron microscopy, but can be collected with considerably greater throughput, enabling examination of a broad range of experimental conditions. Using the tethered vesicle approach, we have recently found that membrane-bound intrinsically disordered proteins are surprisingly potent drivers of membrane fission. The capacity to drive fission arises from steric pressure generated when disordered domains with large hydrodynamic radii bind to membranes at high local densities. More broadly, the experimental tools described here have the potential to improve our mechanistic understanding of membrane fission in diverse biophysical contexts.

Synergy between intrinsically disordered domains and structured proteins amplifies membrane curvature sensing.

The ability of proteins to sense membrane curvature is essential to cellular function. All known sensing mechanisms rely on protein domains with specific structural features such as wedge-like amphipathic helices and crescent-shaped BAR domains. Yet many proteins that contain these domains also contain large intrinsically disordered regions. Here we report that disordered domains are themselves potent sensors of membrane curvature. Comparison of Monte Carlo simulations with in vitro and live-cell measurements demonstrates that the polymer-like behavior of disordered domains found in endocytic proteins drives them to partition preferentially to convex membrane surfaces, which place fewer geometric constraints on their conformational entropy. Further, proteins containing both structured curvature sensors and disordered regions are more than twice as curvature sensitive as their respective structured domains alone. These findings demonstrate an entropic mechanism of curvature sensing that is independent of protein structure and illustrate how structured and disordered domains can synergistically enhance curvature sensitivity.

Structure Versus Stochasticity-The Role of Molecular Crowding and Intrinsic Disorder in Membrane Fission.

Cellular membranes must undergo remodeling to facilitate critical functions including membrane trafficking, organelle biogenesis, and cell division. An essential step in membrane remodeling is membrane fission, in which an initially continuous membrane surface is divided into multiple, separate compartments. The established view has been that membrane fission requires proteins with conserved structural features such as helical scaffolds, hydrophobic insertions, and polymerized assemblies. In this review, we discuss these structure-based fission mechanisms and highlight recent findings from several groups that support an alternative, structure-independent mechanism of membrane fission. This mechanism relies on lateral collisions among crowded, membrane-bound proteins to generate sufficient steric pressure to drive membrane vesiculation. As a stochastic process, this mechanism contrasts with the paradigm that deterministic protein structures are required to drive fission, raising the prospect that many more proteins may participate in fission than previously thought. Paradoxically, our recent work suggests that intrinsically disordered domains may be among the most potent drivers of membrane fission, owing to their large hydrodynamic radii and substantial chain entropy. This stochastic view of fission also suggests new roles for the structure-based fission proteins. Specifically, we hypothesize that in addition to driving fission directly, the canonical fission machines may facilitate the enrichment and organization of bulky disordered protein domains in order to promote membrane fission by locally amplifying protein crowding.

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.