Kinetic trapping organizes actin filaments within liquid-like protein droplets.

Several actin-binding proteins (ABPs) phase separate to form condensates capable of curating the actin network shapes. Here, we use computational modeling to understand the principles of actin network organization within VASP condensate droplets. Our simulations reveal that the different actin shapes, namely shells, rings, and mixture states are highly dependent on the kinetics of VASP-actin interactions, suggesting that they arise from kinetic trapping. Specifically, we show that reducing the residence time of VASP on actin filaments reduces degree of bundling, thereby promoting assembly of shells rather than rings. We validate the model predictions experimentally using a VASP-mutant with decreased bundling capability. Finally, we investigate the ring opening within deformed droplets and found that the sphere-to-ellipsoid transition is favored under a wide range of filament lengths while the ellipsoid-to-rod transition is only permitted when filaments have a specific range of lengths. Our findings highlight key mechanisms of actin organization within phase-separated ABPs.

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.

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.

Transmembrane coupling of liquid-like protein condensates.

Liquid-liquid phase separation of proteins occurs on both surfaces of cellular membranes during diverse physiological processes. In vitro reconstitution could provide insight into the mechanisms underlying these events. However, most existing reconstitution techniques provide access to only one membrane surface, making it difficult to probe transmembrane phenomena. To study protein phase separation simultaneously on both membrane surfaces, we developed an array of freestanding planar lipid membranes. Interestingly, we observed that liquid-like protein condensates on one side of the membrane colocalized with those on the other side, resulting in transmembrane coupling. Our results, based on lipid probe partitioning and mobility of lipids, suggest that protein condensates locally reorganize membrane lipids, a process which could be explained by multiple effects. These findings suggest a mechanism by which signals originating on one side of a biological membrane, triggered by protein phase separation, can be transferred to the opposite side.

Selective Endocytic Uptake of Targeted Liposomes Occurs within a Narrow Range of Liposome Diameters.

Cell surface receptors facilitate signaling and nutrient uptake. These processes are dynamic, requiring receptors to be actively recycled by endocytosis. Due to their differential expression in disease states, receptors are often the target of drug-carrier particles, which are adorned with ligands that bind specifically to receptors. These targeted particles are taken into the cell by multiple routes of internalization, where the best-characterized pathway is clathrin-mediated endocytosis. Most studies of particle uptake have utilized bulk assays rather than observing individual endocytic events. As a result, the detailed mechanisms of particle uptake remain obscure. To address this gap, we employed a live-cell imaging approach to study the uptake of individual liposomes as they interact with clathrin-coated structures. By tracking individual internalization events, we find that the size of liposomes rather than the density of the ligands on their surfaces primarily determines their probability of uptake. Interestingly, targeting has the greatest impact on endocytosis of liposomes of intermediate diameters, with the smallest and largest liposomes being internalized or excluded, respectively, regardless of whether they are targeted. These findings, which highlight a previously unexplored limitation of targeted delivery, can be used to design more effective drug carriers.

Ubiquitin-driven protein condensation promotes clathrin-mediated endocytosis.

Clathrin-mediated endocytosis is an essential cellular pathway that enables signaling and recycling of transmembrane proteins and lipids. During endocytosis, dozens of cytosolic proteins come together at the plasma membrane, assembling into a highly interconnected network that drives endocytic vesicle biogenesis. Recently, multiple labs have reported that early endocytic proteins form liquid-like condensates, which provide a flexible platform for the efficient assembly of endocytic vesicles. Given the importance of this network in the dynamics of endocytosis, how might cells regulate its stability? Many receptors and endocytic proteins are ubiquitylated, while early endocytic proteins such as Eps15 contain ubiquitin-interacting motifs. Therefore, we examined the influence of ubiquitin on the stability of the early endocytic protein network. In vitro, we found that recruitment of small amounts of polyubiquitin dramatically increased the stability of Eps15 condensates, suggesting that ubiquitylation could nucleate endocytic sites. In live cell imaging experiments, a version of Eps15 that lacked the ubiquitin-interacting motif failed to rescue defects in endocytic initiation created by Eps15 knockout. Furthermore, fusion of Eps15 to a deubiquitinase enzyme destabilized nascent endocytic sites within minutes. These results suggest that ubiquitylation drives assembly of the flexible protein network responsible for catalyzing endocytic events. More broadly, this work illustrates a biophysical mechanism by which ubiquitylated transmembrane proteins at the plasma membrane could regulate the efficiency of endocytic recycling.

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 versus becoming 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.

Selective endocytic uptake of targeted liposomes occurs within a narrow range of liposome diameter.

Cell surface receptors facilitate signaling and nutrient uptake. These processes are dynamic, requiring receptors to be actively recycled by endocytosis. Due to their differential expression in disease states, receptors are often the target of drug-carrier particles, which are adorned with ligands that bind specifically to receptors. These targeted particles are taken into the cell by multiple routes of internalization, where the best-characterized pathway is clathrin-mediated endocytosis. Most studies of particle uptake have utilized bulk assays, rather than observing individual endocytic events. As a result, the detailed mechanisms of particle uptake remain obscure. To address this gap, we have employed a live-cell imaging approach to study the uptake of individual liposomes as they interact with clathrin-coated structures. By tracking individual internalization events, we find that the size of liposomes, rather than the density of the ligands on their surfaces, primarily determines their probability of uptake. Interestingly, targeting has the greatest impact on endocytosis of liposomes of intermediate diameters, with the smallest and largest liposomes being internalized or excluded, respectively, regardless of whether they are targeted. These findings, which highlight a previously unexplored limitation of targeted delivery, can be used to design more effective drug carriers.

The ins and outs of membrane bending by intrinsically disordered proteins.

Membrane curvature is essential to diverse cellular functions. While classically attributed to structured domains, recent work illustrates that intrinsically disordered proteins are also potent drivers of membrane bending. Specifically, repulsive interactions among disordered domains drive convex bending, while attractive interactions drive concave bending, creating membrane-bound, liquid-like condensates. How might disordered domains that contain both repulsive and attractive domains affect curvature? Here, we examined chimeras that combined attractive and repulsive interactions. When the attractive domain was closer to the membrane, its condensation amplified steric pressure among repulsive domains, leading to convex curvature. In contrast, when the repulsive domain was closer to the membrane, attractive interactions dominated, resulting in concave curvature. Further, a transition from convex to concave curvature occurred with increasing ionic strength, which reduced repulsion while enhancing condensation. In agreement with a simple mechanical model, these results illustrate a set of design rules for membrane bending by disordered proteins.

Fibril formation and ordering of disordered FUS LC driven by hydrophobic interactions.

Biomolecular condensates, protein-rich and dynamic membrane-less organelles, play critical roles in a range of subcellular processes, including membrane trafficking and transcriptional regulation. However, aberrant phase transitions of intrinsically disordered proteins in biomolecular condensates can lead to the formation of irreversible fibrils and aggregates that are linked to neurodegenerative diseases. Despite the implications, the interactions underlying such transitions remain obscure. Here we investigate the role of hydrophobic interactions by studying the low-complexity domain of the disordered 'fused in sarcoma' (FUS) protein at the air/water interface. Using surface-specific microscopic and spectroscopic techniques, we find that a hydrophobic interface drives fibril formation and molecular ordering of FUS, resulting in solid-like film formation. This phase transition occurs at 600-fold lower FUS concentration than required for the canonical FUS low-complexity liquid droplet formation in bulk. These observations highlight the importance of hydrophobic effects for protein phase separation and suggest that interfacial properties drive distinct protein phase-separated structures.

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.

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.

Liquid-like VASP condensates drive actin polymerization and dynamic bundling.

The organization of actin filaments into bundles is required for cellular processes such as motility, morphogenesis, and cell division. Filament bundling is controlled by a network of actin-binding proteins. Recently, several proteins that comprise this network have been found to undergo liquid-liquid phase separation. How might liquid-like condensates contribute to filament bundling? Here, we show that the processive actin polymerase and bundling protein, VASP, forms liquid-like droplets under physiological conditions. As actin polymerizes within VASP droplets, elongating filaments partition to the edges of the droplet to minimize filament curvature, forming an actin-rich ring within the droplet. The rigidity of this ring is balanced by the droplet's surface tension, as predicted by a continuum-scale computational model. However, as actin polymerizes and the ring grows thicker, its rigidity increases and eventually overcomes the surface tension of the droplet, deforming into a linear bundle. The resulting bundles contain long, parallel actin filaments that grow from their tips. Significantly, the fluid nature of the droplets is critical for bundling, as more solid droplets resist deformation, preventing filaments from rearranging to form bundles. Once the parallel arrangement of filaments is created within a VASP droplet, it propagates through the addition of new actin monomers to achieve a length that is many times greater than the initial droplet. This droplet-based mechanism of bundling may be relevant to the assembly of cellular architectures rich in parallel actin filaments, such as filopodia, stress fibers, and focal adhesions.

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.

Clathrin: Bender or bystander?

Cell biologists have long debated the role of clathrin in curving membranes during endocytosis. New findings from Cail et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202109013) take an innovative approach to directly demonstrate the indispensable functions of both clathrin and its adaptor network in shaping endocytic vesicles.

Gap Junction-Mediated Delivery of Polymeric Macromolecules.

Cellular delivery of therapeutic macromolecules such as proteins, peptides, and nucleic acids remains limited due to inefficient transport across the cellular plasma membrane. Gap junction channels, composed of connexin proteins, provide a mechanism for direct transfer of small molecules across membranes, and recent evidence suggests that the transfer of larger, polymer-like molecules such as microRNAs may be possible. Here, we report direct evidence of gap junction-mediated transfer of polymeric macromolecules. Specifically, we examined the transport of dextran chains with molecular weights ranging from 10 to 70 kDa. We found that dextran chains of up to 40 kDa can diffuse through at least five cell layers in a gap junction-dependent manner within a 30 min time frame. Further, we evaluated the ability of connectosomes, cell-derived vesicles containing functional connexin proteins, to be loaded with dextran chains. By opening connexon hemichannel pores within the membranes of connectosomes, we found that 10 kDa dextran was loaded into more than 90% of vesicles, with reduced levels of loading for dextran chains of larger molecular weight. Upon delivering 10 kDa dextran-loaded connectosomes to cells, we further found that connectosomes transferred these membrane-impermeable molecules to the cellular cytosol with dramatically improved efficiency in comparison to the delivery of free, unencapsulated dextran. Collectively, these results reveal that polymeric macromolecules can be delivered to cells via gap junctions, suggesting that the gap junction route may be useful for the delivery of polymeric therapeutic molecules, such as nucleic acids and peptides.

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.

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.

Molecular tension in syndecan-1 is regulated by extracellular mechanical cues and fluidic shear stress.

The endothelium plays a central role in regulating vascular homeostasis and is key in determining the response to materials implanted in the vascular system. Endothelial cells are uniquely sensitive to biophysical cues from applied forces and their local cellular microenvironment. The glycocalyx is a layer of proteoglycans, glycoproteins and glycosaminoglycans that lines the luminal surface of the vascular endothelium, interacting directly with the components of the blood and the forces of blood flow. In this work, we examined the changes in mechanical tension of syndecan-1, a cell surface proteoglycan that is an integral part of the glycocalyx, in response to substrate stiffness and fluidic shear stress. Our studies demonstrate that syndecan-1 has higher mechanical tension in regions of cell adhesion, on and in response to nanotopographical cues. In addition, we found that substrate stiffness also regulated the mechanical tension of syndecan-1 and altered its binding to actin, myosin iiB and signaling intermediates including Src, PKA and FAK. Application of fluidic shear stress created a gradient in tension in syndecan-1 and led to enhanced association with actin, Src, myosin IIb and other cytoskeleton related molecules. Overall, our studies support that syndecan-1 is responsive to the mechanical environment of the cells and alters its association with actin and signaling intermediates in response to mechanical stimuli.