Biology subjects

Lafer, E. M.

Publications and source records attributed to Lafer, E. M..

14 recordsLinked to original sources

bioRxiv2026

Endocytic accessory proteins assemble clathrin while simultaneously destabilizing protein condensates

During endocytosis a dynamic protein network is responsible for assembly of the clathrin coat. Early in the process the Eps15 protein is thought to undergo liquid-liquid phase-separation to form biomolecular condensates. These condensates facilitate clathrin assembly, producing clathrin-coated vesicles that are ultimately excluded from the condensate as the vesicles depart from the plasma membrane. An array of distinct clathrin accessory proteins drive this process, yet their interactions with protein condensates have not been explored. Here we show that clathrin accessory proteins promote the assembly of the clathrin lattice by simultaneously destabilizing protein condensates. By adding diverse accessory proteins to Eps15 condensates, we observed that accessory proteins which cross-linked Eps15 proteins stabilized condensates, while accessory proteins that compete with Eps15-Eps15 interactions destabilized condensates. Interestingly, accessory proteins that destabilized condensates also promoted the assembly of clathrin and its exclusion from condensates. In contrast, accessory proteins that stabilized condensates opposed exclusion of clathrin. Finally, we demonstrated that decreasing condensate stability enhanced clathrin assembly and exclusion, even when no additional clathrin assembly motifs were introduced. Taken together, these results help to explain how condensates can catalyze the assembly of coated vesicles by providing both a substrate for their initiation and a driving force for their exclusion and ultimate departure. Statement of SignificanceThis work is significant because it reveals how biomolecular condensates could actively drive, rather than merely host, clathrin-mediated endocytosis. By showing that clathrin accessory proteins can tune condensate stability to simultaneously promote clathrin lattice assembly and exclusion, this study uncovers a physical mechanism that could link phase separation to membrane trafficking. The finding that condensate destabilization alone enhances clathrin exclusion reframes condensates as dynamic regulators that could provide both an initiation platform and a force for vesicle maturation and release. More broadly, this work advances our understanding of how cells coordinate assembly and disassembly of complex macromolecular structures through regulated changes in condensate material properties.

biophysics↗

bioRxiv2026

Intrinsically disordered ligands for the control of receptor uptake by endocytosis

Endocytosis plays a crucial role in signaling, recycling, and degradation of receptors. Controlling endocytosis of specific receptors is therefore a major goal for both basic science and medicine. While antibody-induced dimerization can drive signaling-induced uptake of some receptors, the steric bulk of antibodies generally inhibits endocytosis, such that control over receptor uptake remains an unmet need. Recent work has demonstrated that attractive interactions between intrinsically disordered proteins drive inward membrane curvature, a key step in endocytosis. To harness this phenomenon for the control of receptor uptake, we designed chimeras that consisted of disordered domains fused to ligands for specific receptors. These chimeras condensed on membrane surfaces, driving receptor clustering and uptake via clathrin-mediated endocytosis. In contrast, chimeras that repelled one another resisted condensation, helping receptors escape endocytosis and remain on the cell surface. Taken together, these results suggest that by modulating the amino acid sequence of intrinsically disordered ligands, we can promote or hinder the internalization of specific receptors by endocytic pathways. More broadly, these findings suggest a generalizable strategy for controlling the plasma membrane lifetime of diverse receptors, opening up new pathways for modulating cellular behavior and delivering therapeutics. Significance StatementCells regulate signaling by continuously internalizing receptors from their surfaces using endocytosis. Controlling receptor internalization would provide new tools for addressing diverse diseases. While antibodies can cluster specific receptors on the cell surface, their steric bulk and rigidity inhibit endocytosis. In contrast, here we demonstrate that engineered ligands containing intrinsically disordered domains form flexible complexes that precisely control receptor internalization. Disordered ligands that attract one another condense receptors at sites of endocytosis, driving uptake, while repulsive disordered ligands prevent condensation such that receptors remain on the cell surface. By tuning the amino acid sequence of the disordered domain, a ligand can be switched from promoting to inhibiting receptor internalization, offering the opportunity to control cell signaling and therapeutic delivery.

biophysics↗

bioRxiv2025

A balance between nucleating and elongating actin filaments controls deformation of protein condensates

Protein condensates use multivalent binding and surface tension to assemble actin filaments into diverse architectures, reminiscent of filopodia and stress fibers. During this process, nucleation of new filaments and elongation of existing filaments inherently compete for a shared pool of actin monomers. Here we show that a balance between these competing processes is required to deform condensates of VASP, an actin binding protein, into structures with high aspect ratios. Addition of magnesium, which promotes filament nucleation, helped actin to deform condensates into high aspect ratio structures. In contrast, addition of profilin, which inhibits filament nucleation, allowing existing filaments to elongate, caused actin to assemble into ring-like bundles that failed to substantially increase condensate aspect ratio. Computational modeling helped to explain these results by predicting that a group of short linear filaments, which can apply asymmetric pressure to the condensate boundary, is needed to increase condensate aspect ratio. In contrast, a small number of long filaments with the same total actin content should fail to overcome the droplet surface tension, forming a ring-like bundle. To test these predictions, we introduced gelsolin, which severed log filaments within rings, creating new barbed ends. The resulting set of shorter filaments regained the ability to deform condensates into high aspect ratio structures. Collectively, these results suggest that a balance of actin filament nucleation and elongation is required to deform protein condensates. More broadly, these findings illustrate how protein condensates can balance multiple kinetic processes to direct the assembly of diverse cytoskeletal architectures.

biophysics↗

bioRxiv2025

Of condensates and coats - reciprocal regulation of clathrin assembly and the growth of protein networks

Clathrin-mediated endocytosis is essential for membrane traffic, impacting a diverse range of cellular processes including cell signaling homeostasis, cell adhesion, and receptor recycling. During endocytosis, invagination of the plasma membrane is coordinated by a network of proteins that recruit and assemble the clathrin coat. Recent work demonstrated that clathrin accessory proteins which arrive early at endocytic sites, such as Eps15 and Fcho2, form phase-separated condensates that recruit downstream machinery, promoting assembly and maturation of clathrin-coated vesicles. However, the mechanisms by which protein condensates regulate - and are regulated by - clathrin assembly remain unclear. Using in vitro reconstitution and nuclear magnetic resonance spectroscopy, we demonstrate that protein condensates provide a platform for recruitment and assembly of clathrin triskelia. This condensate driven assembly is enhanced in the presence of the accessory protein, AP2, which is readily incorporated within condensates. In turn, clathrin assembly restricted the growth of condensates, exhibiting surfactant-like behavior that stabilized protein-protein interactions while imposing the preferred curvature of the clathrin lattice. This mutual regulation promotes efficient assembly of clathrin-coated vesicles while preventing uncontrolled expansion of protein condensates. More broadly, reciprocal regulation of protein condensates and clathrin coats may provide a framework for understanding how intrinsically disordered and structured protein assemblies can work together to build complex cellular architectures.

biophysics↗

bioRxiv2025

Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis

Clathrin-mediated endocytosis internalizes proteins and lipids from the cell surface, supporting nutrient uptake, signaling, and membrane trafficking. Recent work has demonstrated that a flexible, liquid-like network of initiator proteins is responsible for catalyzing assembly of clathrin-coated vesicles in diverse organisms including yeast, mammals, and plants. How do cells regulate the assembly of this dynamic network to produce cargo-loaded vesicles? Here we reveal the ability of an endocytic adaptor protein, Epsin1, to conditionally stabilize the initiator protein network, creating a cargo-dependent checkpoint during clathrin-mediated endocytosis. Epsin1 is known to recruit ubiquitylated transmembrane proteins to endocytic sites. Using in vitro assays, we demonstrate that Epsin1 uses competitive binding and steric repulsion to destabilize condensation of initiator proteins in the absence of ubiquitin. However, when polyubiquitin is present, Epsin1 binds to both ubiquitin and initiator proteins, creating attractive interactions that stabilize condensation. Similarly, in mammalian cells, endocytic dynamics and ligand uptake are disrupted by removal of either ubiquitin or Epsin1. Surprisingly, when Epsin1 and ubiquitin are removed simultaneously, endocytic defects are rescued to near wildtype levels, although endocytic sites lose the ability to distinguish between ubiquitylated and non-ubiquitylated cargos. Taken together, these results suggest that Epsin1 tunes protein condensation to ensure the presence of ubiquitylated cargo during assembly of clathrin-coated vesicles. More broadly, these findings illustrate how a balance of attractive and repulsive molecular interactions controls the stability of liquid-like protein networks, providing dynamic control over key cellular events.

cell biology↗

bioRxiv2024

Liquid-like condensates that bind actin can polymerize and bundle actin filaments

Liquid-like protein condensates perform diverse physiological functions. Previous work showed that VASP, a processive actin polymerase, forms condensates that polymerize and bundle actin. To minimize their curvature, filaments accumulated at the inner condensate surface, ultimately deforming the condensate into a rod-like shape, filled with a bundle of parallel filaments. Here we show that this behavior does not require proteins with specific polymerase activity. Specifically, we found that condensates composed of Lamellipodin, a protein that binds actin but is not an actin polymerase, were also capable of polymerizing and bundling actin filaments. To probe the minimum requirements for condensate-mediated actin bundling, we developed an agent-based computational model. Guided by its predictions, we hypothesized that any condensate-forming protein that binds actin could bundle filaments through multivalent crosslinking. To test this idea, we added an actin-binding motif to Eps15, a condensate-forming protein that does not normally bind actin. The resulting chimera formed condensates that drove efficient actin polymerization and bundling. Collectively, these findings broaden the family of proteins that could organize cytoskeletal filaments to include any actin-binding protein that participates in protein condensation.

biophysics↗

bioRxiv2023

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 groups have reported that early endocytic proteins form flexible condensates, which provide a platform for 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 assemblies. 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 deubiquitylase enzyme destabilized nascent endocytic sites within minutes. In both in vitro and live cell settings, dynamic exchange of Eps15 proteins, a hallmark of liquid-like systems, was modulated by Eps15-Ub interactions. These results collectively 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. Significance StatementThe assembly of proteins into dynamic, liquid-like condensates is an emerging principle of cellular organization. During clathrin-mediated endocytosis, a liquid-like protein network catalyzes vesicle assembly. How do cells regulate these assemblies? Here we show that ubiquitin and endocytic proteins form a dynamic, mutually-reinforcing protein network in vitro and in live cells. To probe the impact of ubiquitylation on the dynamics of endocytosis, we engineered opto-genetic control over recruitment of proteins to nascent endocytic sites. While recruitment of wildtype proteins promoted endocytosis, recruitment of deubiquitylases, enzymes capable of removing ubiquitin, resulted in disassembly of endocytic sites within minutes. These results illustrate that ubiquitylation can regulate the fate of endocytic structures, elucidating a functional connection between protein condensates, endocytosis, and ubiquitin signaling.

cell biology↗

bioRxiv2023

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 restricting diffusion to a two-dimensional surface, lipid bilayers can nucleate phase separation at far lower concentrations compared to 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 model disordered domains, FUS LC and LAF1-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 LAF1-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.

biophysics↗

bioRxiv2023

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. SIGNIFICANCE STATEMENTReorganization of actin filaments allows cells to migrate, which is required for embryonic development, wound healing, and cancer metastasis. During migration, the leading-edge of the cell consists of needle-like protrusions of bundled actin, which emanate from a sheet of branched actin. Given that the proteins responsible for both architectures are present simultaneously, what determines whether actin filaments will be branched or bundled? Here we show that liquid-like condensates, composed of both branching and bundling proteins, can mediate the inherent competition between these fundamentally different ways of organizing actin networks. This work demonstrates that by tuning the composition of condensates, we can recapitulate the transition from branched to bundled networks, a key step in cell migration.

biophysics↗

bioRxiv2022

Liquid-like assembly of VASP drives actin polymerization and 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 droplets 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.

biophysics↗

bioRxiv2022

Molecular Mechanisms of Steric Pressure Generation and Membrane Remodeling by Intrinsically 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 Forster 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.

biophysics↗

bioRxiv2020

Clathrin senses membrane curvature

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.

molecular biology↗

bioRxiv2019

Liquid-like protein interactions catalyze assembly of endocytic vesicles

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.

cell biology↗

bioRxiv2019

Hsc70 Ameliorates the Vesicle Recycling Defects Caused by Excess -Synuclein at Synapses

-Synuclein overexpression and aggregation are linked to Parkinsons disease (PD), dementia with Lewy bodies (DLB), and several other neurodegenerative disorders. In addition to effects in the cell body, -synuclein accumulation occurs at presynapses where the protein is normally localized. While it is generally agreed that excess -synuclein impairs synaptic vesicle trafficking, the underlying mechanisms are unknown. We show here that acute introduction of excess human -synuclein at a classic vertebrate synapse, the lamprey reticulospinal synapse, selectively impaired the uncoating of clathrin-coated vesicles (CCVs) during synaptic vesicle recycling, leading to a severe depletion of synaptic vesicles. Furthermore, human -synuclein and lamprey {gamma}-synuclein both interact in vitro with Hsc70, the chaperone protein that uncoats CCVs at synapses. After introducing excess -synuclein to lamprey axons, Hsc70 availability was reduced at the synapses, suggesting Hsc70 sequestration as a possible mechanism underlying the synaptic vesicle trafficking defects. In support of this hypothesis, increasing the levels of exogenous Hsc70 together with -synuclein ameliorated the CCV uncoating and vesicle recycling defects. These experiments identify a reduction in Hsc70 availability at synapses, and consequently its function, as the mechanism by which -synuclein induces synaptic vesicle recycling defects. To our knowledge, this is the first report of a viable chaperone-based strategy for reversing the toxic impacts of excess -synuclein at synapses, which may be of value for ameliorating synaptic defects in PD and other synuclein-linked diseases.\n\nSIGNIFICANCE STATEMENTSynaptic defects caused by -synuclein overexpression are linked to cognitive deficits in PD and other diseases. However, the mechanisms by which excess -synuclein impairs synaptic vesicle trafficking are unknown. Data presented here demonstrate that acute introduction of excess -synuclein at a classical vertebrate synapse selectively inhibits CCV uncoating, leading to impaired vesicle recycling. Furthermore, increasing -synuclein reduced synaptic levels of Hsc70, the clathrin uncoating ATPase. Subsequently increasing Hsc70 restored CCV uncoating and improved vesicle recycling. This study identifies a novel molecular mechanism underlying the -synuclein-induced synaptic defects and presents one viable strategy for reversing them.

neuroscience↗