Biology subjects

Hayden, C. C.

Publications and source records attributed to Hayden, C. C..

8 recordsLinked to original sources

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↗

bioRxiv2024

Poly-ubiquitylated transmembrane proteins outcompete other cargo for limited space inside clathrin-coated vesicles

Endocytic recycling of transmembrane proteins is essential to cell signaling, ligand uptake, protein traffic and degradation. The intracellular domains of many transmembrane proteins are ubiquitylated, which promotes their internalization by clathrin-mediated endocytosis. How might this enhanced internalization impact endocytic uptake of transmembrane proteins that lack ubiquitylation? Recent work demonstrates that diverse transmembrane proteins compete for space within highly crowded endocytic structures, suggesting that enhanced internalization of one group of transmembrane proteins may come at the expense of other groups. Here we show that preferential internalization of poly-ubiquitylated transmembrane proteins results in reduced endocytosis of mono-ubiquitylated and non-ubiquitylated proteins. Using a combination of live-cell imaging and ligand uptake assays, we confirmed that increased ubiquitylation correlates with increased internalization by clathrin-coated vesicles. Further, poly-ubiquitylated receptors significantly outcompeted their mono-ubiquitylated and non-ubiquitylated counterparts for localization to endocytic sites and uptake of extracellular ligands. These findings demonstrate the inherent interdependence of transmembrane protein recycling, suggesting that clathrin-coated vesicles act as selective filters, prioritizing highly ubiquitylated transmembrane proteins for uptake while leaving proteins with little or no ubiquitylation behind. Given that poly-ubiquitylation is thought to signal protein aging and damage, our findings suggest a mechanism for selective internalization of high priority cargo proteins, with simultaneously exclusion and protection of functional proteins that lack poly-ubiquitylation.

cell biology↗

bioRxiv2024

A live cell imaging-based assay for tracking particle uptake by clathrin-mediated endocytosis

A popular strategy for therapeutic delivery to cells and tissues is to encapsulate therapeutics inside particles that cells internalize via endocytosis. The efficacy of particle uptake by endocytosis is often studied in bulk using flow cytometry and Western blot analysis and confirmed using confocal microscopy. However, these techniques do not reveal the detailed dynamics of particle internalization and how the inherent heterogeneity of many types of particles may impact their endocytic uptake. Toward addressing these gaps, here we present a live-cell imaging-based method that utilizes total internal reflection fluorescence (TIRF) microscopy to track the uptake of a large ensemble of individual particles in parallel, as they interact with the cellular endocytic machinery. To analyze the resulting data, we employ an open-source tracking algorithm in combination with custom data filters. This analysis reveals the dynamic interactions between particles and endocytic structures, which determine the probability of particle uptake. In particular, our approach can be used to examine how variations in the physical properties of particles (size, targeting, rigidity), as well as heterogeneity within the particle population, impact endocytic uptake. These data impact the design of particles toward more selective and efficient delivery of therapeutics to cells.

biophysics↗

bioRxiv2023

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.

bioengineering↗

bioRxiv2022

Lateral compression of lipids drives transbilayer coupling of liquid-like protein condensates

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

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↗