Biology subjects

Owen, D. J.

Publications and source records attributed to Owen, D. J..

10 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↗

bioRxiv2025

The mechanistic basis of cargo selection during Golgi maturation

The multiple cisternae of the Golgi apparatus contain resident membrane proteins crucial for lipid and protein glycosylation. How Golgi residents remain in their designated compartments despite a constant flow of secretory cargo is incompletely understood. Here, we determined the structure of the COPI vesicle coat containing GOLPH3, an adaptor protein that binds the cytosolic tails of many Golgi residents. The structure, together with structure-guided mutagenesis and functional assays, reveals how GOLPH3 uses coincidence detection of COPI and lipid to engage Golgi residents preferentially at late cisternae. Our findings rationalize the logic of cisternal maturation and explain how COPI can engage different types of substrates in different Golgi cisternae to retrieve some proteins back to the ER while retaining others within the Golgi apparatus.

cell biology↗

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

RABGAP1 acts as a sensor to facilitate sorting and processing of amyloid precursor protein

A key hallmark of Alzheimers disease (AD) is the accumulation of extracellular amyloid-{beta} plaques in the brains of patients. Amyloid-{beta} is a 40-42 amino acid peptide produced by the proteolytic processing of amyloid precursor protein (APP) by a series of membrane-bound proteases. APP is a type-I transmembrane protein and thus its trafficking to encounter the proteases represents a rate-limiting step in the progression of AD. Although there has been a focused research effort to understand APP processing, its trafficking itinerary and machinery is incompletely understood. To address this we have performed an unbiased interaction screen for interactors of the cytosolic tail of APP. We identified previously characterised APP binders, as well as novel interactors. We have mapped the binding of APP to multiple new machineries, including RABGAP1. We have demonstrated that RAB-GAP1 partially co-localises with APP and directly interacts with a YENPTY motif in the APP cytosolic tail. Depletion or overexpression of RABGAP1 caused mistrafficking and misprocessing of endogenous APP in both human and rodent neurons. Interestingly, this effect was dependent on the GAP activity of RABGAP1, demonstrating that RABGAP1 affects the trafficking of APP by modulating the RAB activity on endosomal subdomains. This novel trafficking mechanism has implications for other NPXY cargoes and also presents a possible therapeutic avenue to explore.

cell biology↗

bioRxiv2024

Combined thermodynamic and time-resolved structural analysis of interactions between AP2 and biomimetic plasma membranes provides insights into clathrin-mediated endocytosis

Clathrin mediated endocytosis (CME) is the main mechanism for swift and selective uptake of proteins into eukaryotic cells. CME is initiated by recruitment to the plasma membrane (PM) of the adaptor protein AP2, which recognizes the PM-associated lipid PtdIns(4,5)P2, as well as the protein cargo to be internalized. Nonetheless, many aspects of this process remain unclear due to their in vivo complexity. Here, a thermodynamic and time-resolved structural analysis of AP2 binding to different biomimetic PM was undertaken under physiological conditions using a combination of neutron reflectometry, interfacial tensiometry and rheology, and atomic force microscopy. The resultant in vitro data replicated previous in vivo observations, as well as yielded biophysical insights into normal and aborted CME. The presence of cargo may not be pivotal for the "activating" conformational change of AP2. However, the presence of cargo extends AP2s residence time on the membrane surface, due to slower on- and off-rates, thereby tentatively giving sufficient time for CME to proceed fully. Moreover, upon interaction with AP2, phospholipid lateral diffusion decreases markedly, inducing a gel phase attributed to creating a percolated network involving AP2 on the membrane, which could potentially serve as a mechanism for modulating subsequent clathrin binding.

biophysics↗

bioRxiv2023

Cargo selective vesicle tethering: the structural basis for binding of specific cargo proteins by the Golgi tether component TBC1D23

For accurate membrane traffic it is essential that vesicles and other carriers tether and fuse to only the correct compartment. The TGN-localised golgins golgin-97 and golgin-245 capture transport vesicles arriving from endosomes via the protein TBC1D23 that forms a bridge between the golgins and endosome-derived vesicles. The C-terminal domain of TBC1D23 is responsible for vesicle capture, but how it recognises a specific type of vesicle was unclear. A search for binding partners of the C-terminal domain surprisingly revealed direct binding to carboxypeptidase D (CPD) and syntaxin-16, both known cargo proteins of the captured vesicles. Binding is via a TLY-containing sequence present in both proteins. A crystal structure reveals how this "acidic TLY motif" binds to the C-terminal domain of TBC1D23. An acidic TLY motif is also present in the tails of other endosome-to-Golgi cargo, and these also bind TBC1D23. Structure-guided mutations in the C-terminal domain that disrupt motif binding in vitro also block vesicle capture in vivo. Thus, TBC1D23 attached to golgin-97 and golgin-245 captures vesicles by a previously undescribed mechanism: the recognition of a motif shared by cargo proteins carried by the vesicle. One sentence summaryA class of transport vesicle destined for the Golgi is recognized by a tether binding directly to the cargo it is carrying.

cell biology↗

bioRxiv2022

FCHO controls AP2's critical endocytic roles through a PtdIns4,5P2 membrane-dependent switch

Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo-adaptor AP2 requires membrane-localised FCHO. Here, live-cell eTIRF-SIM shows that FCHO marks sites of clathrin- coated pit (CCP) initiation, which mature into uniform sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Eps15 ring. We dissect the network of interactions between the FCHO interdomain-linker and AP2, which concentrates, orients, tethers and partially destabilizes closed AP2 at the plasma membrane. AP2s subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with PtdIns4,5P2, clathrin, cargo and CME accessory factors. FCHO can now relocate toward a CCPs outer edge to engage and activate further AP2s to drive CCP growth/maturation. 125 character summaryFCHO primes AP2 for CCV incorporation, a process that triggers FCHO release to enable activation/recruitment of further AP2s

cell biology↗

bioRxiv2020

Architecture and mechanism of metazoan retromer:SNX3 tubular coat assembly

Retromer is a master regulator of cargo retrieval from endosomes, which is critical for many cellular processes including signalling, immunity, neuroprotection and virus infection. To function in different trafficking routes, retromer core (VPS26/VPS29/VPS35) assembles with a range of sorting nexins to generate tubular carriers and incorporate assorted cargoes. We elucidate the structural basis of membrane remodelling and coupled cargo recognition by assembling metazoan and fungal retromer core trimers on cargo-containing membranes with sorting nexin adaptor SNX3 and determining their structures using cryo-electron tomography. Assembly leads to formation of tubular carriers in the absence of canonical membrane curvature drivers. Interfaces in the retromer coat provide a structural explanation for Parkinsons disease-linked mutations. We demonstrate that retromer core trimer forms an invariant, evolutionarily-conserved scaffold that can incorporate different auxiliary membrane adaptors by changing its mode of membrane recruitment, so modulating membrane bending and cargo incorporation and thereby allowing retromer to traffic assorted cargoes along different cellular transport routes.

molecular biology↗

bioRxiv2020

Architecture of the AP2:clathrin coat on the membranes of clathrin-coated vesicles

Clathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cells plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which cargo-selecting clathrin adaptors are attached. AP2 is the key adaptor in CME. Crystallography has shown AP2 to adopt a range of conformations. Here we used cryo-electron microscopy, tomography and subtomogram averaging to determine structures, interactions and arrangements of clathrin and AP2 at the key steps of coat assembly, from AP2 in solution to membrane-assembled clathrin-coated vesicles (CCVs). AP2 binds cargo and PtdIns(4,5)P2-containing membranes via multiple interfaces, undergoing conformational rearrangement from its cytosolic state. The binding mode of AP2 {beta}2-appendage into the clathrin lattice in CCVs and buds implies how the adaptor structurally modulates coat curvature and coat disassembly.

molecular biology↗

bioRxiv2019

Structure of herpes simplex virus pUL7:pUL51, a conserved complex required for efficient herpesvirus assembly

Herpesviruses are an ancient family of highly-prevalent human and animal pathogens that acquire their membrane envelopes in the cytoplasm of infected cells. While multiple conserved viral proteins are known to be required for efficient herpesvirus production, many of these proteins lack identifiable structural homologues and the molecular details of herpesvirus assembly remain unclear. We have characterized the complex of assembly proteins pUL7 and pUL51 from herpes simplex virus (HSV)-1, an -herpesvirus, using multi-angle light scattering and small-angle X-ray scattering with chemical crosslinking. HSV-1 pUL7 and pUL51 form a stable 1:2 complex that is capable of higher-order oligomerization in solution. We solved the crystal structure of this complex, revealing a core heterodimer comprising pUL7 bound to residues 41-125 of pUL51. While pUL7 adopts a previously-unseen compact fold, the extended helix-turn-helix conformation of pUL51 resembles the cellular endosomal complex required for transport (ESCRT)-III component CHMP4B, suggesting a direct role for pUL51 in promoting membrane scission during virus assembly. We demonstrate that the interaction between pUL7 and pUL51 homologues is conserved across human -, {beta}- and {gamma}-herpesviruses, as is their association with trans-Golgi membranes in cultured cells. However, pUL7 and pUL51 homologues do not form complexes with their respective partners from different virus families, suggesting that the molecular details of the interaction interface have diverged. Our results demonstrate that the pUL7:pUL51 complex is conserved across the herpesviruses and provide a structural framework for understanding its role in herpesvirus assembly.\n\nSignificance StatementHerpesviruses are extremely common human pathogens that cause diseases ranging from cold sores to cancer. Herpesvirus acquire their membrane envelope in the cytoplasm via a conserved pathway, the molecular details of which remain unclear. We have solved the structure of a complex between herpes simplex virus (HSV)-1 proteins pUL7 and pUL51, two proteins that are required for efficient HSV-1 assembly. We show that formation of this complex is conserved across distantly-related human herpesviruses, as is the association of these homologues with cellular membranes that are used for virion assembly. While pUL7 adopts a previously-unseen fold, pUL51 resembles key cellular membrane-remodeling complex components, suggesting that the pUL7:pUL51 complex may play a direct role in deforming membranes to promote virion assembly.

microbiology↗